September 2024
Volume 65, Issue 11
Open Access
Retinal Cell Biology  |   September 2024
iPSC-Derived LCHADD Retinal Pigment Epithelial Cells Are Susceptible to Lipid Peroxidation and Rescued by Transfection of a Wildtype AAV-HADHA Vector
Author Affiliations & Notes
  • Tiffany DeVine
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States
  • Gabriela Elizondo
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States
  • Garen Gaston
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States
  • Shannon J. Babcock
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States
  • Dietrich Matern
    Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, United States
  • Mikhail S. Shchepinov
    Institut des Biomolécules Max Mousseron, Université de Montpellier, CNRS, ENSCM, Montpellier, France
    Skolkovo Institute of Science and Technology, Moscow, Russia
  • Mark E. Pennesi
    Ophthalmic Genetics Service, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon, United States
    Retina Foundation of the Southwest, Dallas, Texas, United States
  • Cary O. Harding
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States
  • Melanie B. Gillingham
    Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States
  • Correspondence: Melanie B. Gillingham, Department of Molecular and Medical Genetics, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3098, USA; [email protected]
Investigative Ophthalmology & Visual Science September 2024, Vol.65, 22. doi:https://doi.org/10.1167/iovs.65.11.22
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      Tiffany DeVine, Gabriela Elizondo, Garen Gaston, Shannon J. Babcock, Dietrich Matern, Mikhail S. Shchepinov, Mark E. Pennesi, Cary O. Harding, Melanie B. Gillingham; iPSC-Derived LCHADD Retinal Pigment Epithelial Cells Are Susceptible to Lipid Peroxidation and Rescued by Transfection of a Wildtype AAV-HADHA Vector. Invest. Ophthalmol. Vis. Sci. 2024;65(11):22. https://doi.org/10.1167/iovs.65.11.22.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Progressive choroid and retinal pigment epithelial (RPE) degeneration causing vision loss is a unique characteristic of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a fatty acid oxidation disorder caused by a common c.1528G>C pathogenic variant in HADHA, the α subunit of the mitochondrial trifunctional protein (TFP). We established and characterized an induced pluripotent stem cell (iPSC)-derived RPE cell model from cultured skin fibroblasts of patients with LCHADD and tested whether addition of wildtype (WT) HAHDA could rescue the phenotypes identified in LCHADD-RPE.

Methods: We constructed an rAAV expression vector containing 3′ 3xFLAG-tagged human HADHA cDNA under the transcriptional control of the cytomegalovirus (CMV) enhancer-chicken beta actin (CAG) promoter (CAG-HADHA-3XFLAG). LCHADD-RPE were cultured, matured, and transduced with either AAV-GFP (control) or AAV-HADHA-3XFLAG.

Results: LCHADD-RPE express TFP subunits and accumulate 3-hydroxy-acylcarnitines, cannot oxidize palmitate, and release fewer ketones than WT-RPE. When LCHADD-RPE are exposed to docosahexaenoic acid (DHA), they have increased oxidative stress, lipid peroxidation, decreased viability, and are rescued by antioxidant agents potentially explaining the pathologic mechanism of RPE loss in LCHADD. Transduced LCHADD-RPE expressing a WT copy of TFPα incorporated TFPα-FLAG into the TFP complex in the mitochondria and accumulated significantly less 3-hydroxy-acylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress following DHA exposure than control.

Conclusions: iPSC-derived LCHADD-RPE are susceptible to lipid peroxidation mediated cell death and are rescued by exogenous HADHA delivered with rAAV. These results are promising for AAV-HADHA gene addition therapy as a possible treatment for chorioretinopathy in patients with LCHADD.

Progressive chorioretinopathy associated with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD, OMIM #609016) is a unique complication not seen in any other fatty acid oxidation (FAO) disorder.1 Patients with LCHADD have a c.1528G>C pathogenic variant in HADHA which encodes the α subunit of the mitochondrial trifunctional protein (TFP) while HADHB encodes the β subunit. TFP, a heterotetramer consisting of 2 α- and 2 β-subunits, catalyzes the last three enzymatic steps of mitochondrial long-chain FAO: 2-enoyl-CoA hydratase, LCHAD, and 3-ketoacyl-CoA-thiolase.2 The common c.1528G>C pathogenic variant falls on the LCHAD active site selectively reducing its activity while maintaining the other enzymatic activities of TFP relatively intact.3 This causes a unique accumulation of long-chain 3-hydroxyacylcarnitines, a biomarker for LCHADD. In addition to chorioretinopathy, patients present with hypoketotic hypoglycemia, recurrent rhabdomyolysis crises, cardiomyopathy, and neuropathy.1 The current management for LCHADD consists of a life-long low long-chain fat diet supplemented with medium-chain triglycerides and fasting avoidance.4 Recommendations include monitoring plasma essential fatty acids to guide supplementation in case of deficiency.5 Docosahexaenoic acid (DHA) supplementation in patients with LCHADD with low plasma DHA concentrations improved visual evoked cortical potential (VEP) indicative of an improved visual cycle.6 Early diagnosis and intervention through newborn screening has prevented many disease-associated complications but progression of retinopathy with vision loss has been observed in patients with LCHADD despite early treatment.7,8 
The pathophysiology of LCHADD retinopathy remains incompletely understood. In natural history studies, we have observed that retinal pigment epithelium (RPE) loss is more extensive than photoreceptor loss with progressive stages of chorioretinopathy suggesting that initial RPE loss precedes photoreceptor death.810 Gradual pigment clumping in the macula and decreased fundal autofloresence, indicative of RPE cell involvement, followed by progressive deterioration of the choroid vessels and retina occur in almost all patients with LCHADD.11 Clinically, this manifests as decreased night vision, but progresses to loss of color vision and decreased central vision.9,11,12 However, the cause of RPE death is currently unknown. RPE express all of the FAO enzymes, use fatty acids for energy and create ketones to metabolically support photoreceptors.13 Although it is possible that the loss of energy from FAO compromises the viability of LCHADD-RPE, this chorioretinopathy is not observed among patients with other long-chain FAO disorders, such as very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, for which similar energy deficits are expected. Alternatively, LCHADD chorioretinopathy may be explained by the unique increase in 3-hydroxy fatty acids that may selectively increase oxidative stress in the RPE. 
The RPE has many important functions in the retina: absorption of scattered light, transepithelial transport, ion buffering in the subretinal space, participation in the visual cycle, and photoreceptor outer segments (POS) phagocytosis.14 Phagocytosis of POS, which are enriched in polyunsaturated fatty acids (PUFAs), is an important function of RPE that may be associated with increased oxidative stress.15 POS possess the highest relative concentration of PUFAs compared with other body tissues and the presence of multiple double bonds in PUFAs make them susceptible to lipid peroxidation by reactive oxygen species (ROS).16,17 DHA (C22:6) is a highly unsaturated omega-3 PUFA that accounts for 50% of PUFAs found in the retina.18 DHA is an essential structural component of the cell membrane and it is known to enhance RPE survival in some circumstances.16,18 However, DHA is the most oxidizable fatty acid in humans and can be peroxidized into 4-hydroxyhexenal and 4-hydroxy-7-oxohept-5-enoic acid that lead to the formation of carboxyethylpyrrole, which are all detrimental to RPE and photoreceptors.19 Therefore, oxidative stress due to increased 3-hydroxy fatty acids can potentially cause lipid peroxidation by interacting with DHA in the RPE leading to cell damage and death. 
A publication reported the development of LCHADD-RPE cells derived from patients’ fibroblast through induced pluripotent stem cell (iPSC) technology that were smaller, had disorganized tight junctions with increased melanolysosomes, and accumulated cytosolic neutral lipid droplets.20 Other iPSC-derived RPE have been successfully used to model other retinal disorders.2126 Here, we expand on the initial characterization of iPSC-derived LCHADD-RPE by describing mature patient-specific LCHADD-RPE cells in culture and the evaluation of the pathophysiologic mechanisms of LCHADD-associated RPE cell death. We also demonstrate that the gene addition of a wildtype (WT) copy of HADHA rescued the phenotypes identified in LCHADD-RPE as a potential therapeutic option for LCHADD retinopathy. 
Materials and Methods
iPSC Reprogramming and Culture
Patient fibroblasts were obtained from a skin biopsy conducted to confirm the LCHADD diagnosis in cultured fibroblasts. The patient or a parent/guardian gave informed consent to use stored fibroblasts for research (OHSU eIRB# 9254). They were reprogrammed using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Oct, Sox2, Klf4, and c-Myc) or the Simplicon RNA Reprogramming Kit (OKSG) following the manufacturers’ protocol. Next, we performed single colony subcloning for approximately 10 passages to dilute out the transgenes. RNA was isolated from clones and screened by RT-PCR with primers for either the SEV genome or the OKSG vector backbone. Pluripotency was confirmed by performing immunofluorescence for OCT4, Nanog, and SSEA (Supplementary Fig. S1). Clones were then expanded and cryopreserved. WT cell line (WT001a) was purchased from ATCC. iPSC cells were clump passaged using Versene, grown on Geltrex coated wells and maintained in StemFlex Media. The rock inhibitor Y27632 was added to the media when cells were thawed. 
RPE Differentiation and Culture
iPSC lines were seeded onto a single well from a Geltrex coated plate. Growth factors and small molecules were added to retinal differentiation base media (RDM) consisting of DMEM/F12 with 1 × B27, 1 × N2, 1 × NEAA, as previously described with minor modifications (Supplementary Fig. S2).27,28 On day 14, cells were passaged by adding TrypLE and incubated for 8 minutes at 37°C. The TrypLE was diluted with RDM and passed through a 70 um cell strainer. This was defined as Passage 0 (P0). P0 cells were seeded onto Geltrex coated plates at a density of 105/cm2 and matured in Lonza X-vivo media or RPE maintenance media (RPEM; 1 g/L glucose DMEM, Ham's F-12 nutrient mix, 1X B27 supplement) for 30 days. One week old P0 cells wells were inspected for non-RPE contaminating patches and these were manually removed. After the 30-day maturation period, cells were subjected to rapid passaging where they were split approximately every 7 to 14 days and supplemented with Rock inhibitor Y27632 (10 uM) for 10 days after splitting.29,30 All assays were conducted on P3 or P4 RPE. 
Fatty Acid Oxidation Using the 96-Well Seahorse XFe Analyzer
RPE were seeded onto Geltrex coated 96-well Seahorse plates at a density of 105/cm2 and matured for at least 30 days. The day before the assay, cells were switched to substrate limited media (DMEM 0.5 mM glucose, 1 mM GlutaMAX, 1% FBS, and 1 mM carnitine). The sensor cartridge was hydrated overnight in a non-CO2 chamber. On the day of the assay, medium was exchanged for Krebs Henseleit Buffer (KHB: 111 mM NaCl, 4.7 mM KCl, 2 mM MgSO4, 1.2 mM Na2HPO4, 2.5 mM glucose, 1 mM carnitine, pH 7.4) and incubated for 60 minutes in a non-CO2 chamber. Approximately 15 minutes prior to the start of the assay, BSA (final 0.2%) or BSA-palmitate (final 0.2%/200 uM) was added to appropriate wells. The Seahorse cartridge was preloaded with oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, or FCCP, and Rotenone A. Each genotype (WT or LCHADD) and treatment (BSA or BSA-palmitate) had 3 to 10 replicate wells. Each experiment followed a standard mitochondrial stress test protocol or measured basal oxygen consumption rate (OCR). 
Transepithelial Electrical Resistance
iPSC-RPE were grown on Transwell supports and allowed to mature for 6 weeks. The transepithelial electrical resistance (TEER) was measured using the EVOM3 Volt/Ohm meter. A blank well without cells was measured and subtracted from the sample values. The background adjusted values were then multiplied by the surface area to determine TEER. 
Transmission Electron Microscopy
iPSC-RPE were matured on transmembrane wells and fixed in 2.5% formaldehyde with 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer, pH 7.4 and processed in a Pelco Biowave Pro+ microwave with a SteadyTemp Pro chiller. The membranes were rinsed in buffer three times and then stained with an aqueous solution of 2% osmium tetroxide and 1.5% potassium ferricyanide. Membranes were then stained with 1% uranyl acetate and dehydrated in an increasing ethanol series and fixed in LX112 resin. The membranes were embedded and sectioned. The sections were collected on slot grids and imaged on a Tecnai T12 transmission electron microscope equipped with an AMT Nanosprint12 camera, at 120 kV. 
POS Preparation and Feeding
Bovine rod POS were prepared and conjugated with fluorescein-5-isothiocyanate (FITC), as previously described.27 RPE were seeded in Geltrex coated wells in a chambered coverslip and allowed to mature for 4 weeks. The cells were then stained for rhodopsin and challenged with the FITC labeled POS for 5 hours at 3°C in 5% CO2. The wells were then washed with pre-warmed PBS to remove unbound POS. To determine the level of ROS internalization, an equal volume of 0.4% trypan blue was added to the PBS for 10 minutes to quench extracellular fluorescence. Trypan blue was aspirated, and 100 ul of PBS was added to the well to prevent the cells from drying out. The internalized POS were then documented in fluorescence photomicrographs. Fluorescence intensity was quantified by pixel densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA). 
Ketone Assay
RPE were grown on Transwell supports and allowed to mature for 6 weeks. They were incubated with C16-BSA for 24 hours and β-hydroxybutyrate was measured in the apical chamber (Picoprobe beta Hydrobutyrate Fluorometric Assay Kit; Abcam). 
Triglycerides
RPE cell pellets were processed with the Triglyceride Assay Kit (EnzyChrom, ETGA-200) following manufacturer's instructions. 
Acylcarnitines
RPE were matured for 30 days before incubating with C16-BSA and 1 mM carnitine. Media was then extracted and analyzed by either flow injection analysis tandem mass spectrometry (FIA-MS/MS) similar to plasma samples where media is directly extracted or similar to an in vitro probe assay where the media was spotted onto filter paper, dried, and then extracted and quantified as previously published.31,32 
Immunocytochemistry
RPE were grown on Geltrex coated chambered coverslips for 1 month. To assess lipid peroxidation, cells were stained with C11-BODIPY581/591 (10 uM; Cayman Chemical). To assess oxidative stress, cells were stained with MitoTracker Red CMXRos (100 nM; Invitrogen) for 30 minutes. To assess neutral lipid accumulation, cells were stained with HCS LipidTOX Green Neutral Lipid Stain (Fisher Scientific). For immunofluorescent chemistry (IHC), cells were washed with PBS and fixed with ice-cold 4% paraformaldehyde. Then, the slides were treated with permeabilization solution (PBS, 0.05% Triton X-100) and blocked with PBST buffer (5% goat serum, 0.1% BSA, and 0.1% Triton X-100) for 45 minutes and incubated with indicated primary antibodies overnight at 4°C. The next day, the slides were incubated with the appropriate Alexa Fluor (Invitrogen)-conjugated secondary antibody (goat anti-mouse IgG-Alexa Fluor 488 or goat anti-rabbit IgG-Alexa Fluor 555; 1:400) for 1 hour at room temperature in the dark (antibody concentration details in Supplementary Table S2). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Thermofisher) at 1 ug/mL or Vectashield with DAPI (Vector Labs). Images were captured on a Nikon Eclipse Ts2 Inverted Microscope and analyzed using ImageJ software. 
Western Blots
RPE cell lines were lysed in NP40 lysis buffer supplemented with Complete Mini protease inhibitor cocktail, incubated on ice for 1 hour, and cleared by centrifugation. The 4x Laemmli buffer with 10% β-mercaptoethanol was added to cleared lysate and heated at 98°C for 7 minutes. Proteins were separated on a 10% TGX Stain-Free gels (Bio-Rad) and transferred to a nitrocellulose membrane by a Trans-Blot TurboTransfer System (Bio-Rad). Membranes were then blocked with Tris-buffered saline with 0.1% Tween (TBST) supplemented with 5% milk. The membranes were incubated with the indicated primary antibody overnight at 4°C or for 1 hour at room temperature and matched secondary antibody conjugated to horse-radish peroxidase (HRP; see Supplementary Table S2). The blots were washed in TBST. Bands were visualized using Pico Plus HRP Chemiluminescence kit (Thermo Fisher). The TFPα membrane was re-probed with TFPβ. The membranes were imaged on Azure Sapphire Biomolecular Imager, and total protein was visualized using the Bio-Rad Gel Doc EZ Imager. 
Total Glutathione
Glutathione (GSH) was oxidized by adding 5,5′-dithiobis-(2-nitrobenzoic acid) to RPE resulting in the formation of glutathione disulfide (GSSG) and 5-thio-2-nitrobenzoic acid (TNB). GSSG is then reduced to GSH by glutathione reductase (Sigma-Aldrich) using the reducing equivalent provided by NADPH. The rate of TNB is proportional to the sum of GSH and GSSG present in the cell sample and was determined by spectrophotometry at 412 nm. 
Fatty Acid-BSA Conjugation
Palmitic acid (Cayman Chemical Company), DHA (NU-Check Prep), and dDHA (Gift from Retrotope, Dr. Mikhail S. Shchepinov) were conjugated to fatty-acid free bovine serum albumin (BSA; MP Biomedicals). The fatty acids were dissolved at a 1 mM concentration in pre-warmed 1% BSA at 60°C for approximately 2 hours, mixing frequently. The solution was filtered sterilized and stored at −20°C. Conjugated solution was used at a final concentration of 200 uM fatty acid in 0.2% BSA. 
Cell Viability Assays
RPE cell lines were seeded at approximately 104/cm2 in 96-well plates. Cells were then treated with 200 µM BSA-DHA or 200 uM BSA-dDHA in low glucose media and carnitine (1 mM) for up to 7 days. Specific wells were pretreated with 500 uM NAC (Sigma-Aldrich) or 25 uM Ferrostatin-1 (Cayman Chemical Company). Cells treated with 0.2% BSA were considered controls. After treatment, cells were incubated with Alamar Blue reagent (10% in DMEM no glucose, no phenol) for approximately 4 hours at 37°C. Cell viability was determined by fluorescence (excitation 560/emission 590). At least four technical replicates were analyzed. 
Immunoprecipitation
Mature RPE cell lines were lysed as described for Westerns. After incubating in ice and centrifugation, supernatant was transferred to a new container. Anti-flag coated beads were prepared according to the manufacturer's protocol (ANTI-FLAG M2 Affinity Gel; Sigma-Aldrich). SDS buffer was added and the samples were boiled. IP samples were loaded into the gel with a 5% input in the adjacent well. 
Creation of pAAV-HADHA-3XFLAG
A pAAV-CAG-HADHA-3XFLAG plasmid was created using NEBuilder HiFi DNA Assembly Kit (New England BioLabs) with two fragments that encode HADHA cDNA and three FLAG tags and pAAV-CAG-GFP that had GFP removed using EcoR1-HF and BamHI-HF restriction enzymes. Plasmid was transformed into NEB Stable Competent Escherichia coli (E. coli) cells (New England BioLabs). The final plasmid encoded human HADHA with a chicken beta-actin (CAG) promoter and three FLAG tags attached on the C-terminus with AAV2 ITRs that bookend the construct to allow for packaging into an AAV. Confluent Hek293 cells were transfected using Lipofectamine 2000 (ThermoFisher Scientific) with pAAV2/5 and pAAV-CAG-HADHA-3XFLAG or pAAV-CAG-GFP for 5 days, and the protein was detected by Western blot, and immunofluorescence showed intracellular localization (Supplementary Fig. S5). AAV2/5-HADHA was concentrated using an Amicon Ultra-15 Centrifugal Filter (Millipore Sigma) in 5% sorbitol/PBS and treated with benzoase endonuclease (Millipore Sigma) to clear any unpackaged plasmids. 
Statistics
Experiments consisted of at least three technical repeats. Statistical analysis was performed using GraphPad Prism version 10.2 software. Two-sided unpaired Student t-test, 1-way or 2-way ANOVA with Tukey's analysis for multiple comparisons, and 2-way repeated measures ANOVA with Sidek analysis for multiple comparisons were done when applicable. The P values ≤ 0.05 were considered statistically significant. 
Results
Creation of Patient-Derived iPSC Lines
Two LCHADD patients’ fibroblasts homozygous for the common pathogenic variant, c.1528G>C, in HADHA were reprogrammed into iPSCs (see Supplementary Fig. S1, Supplementary Table S1).33 A WT healthy control iPSC line was purchased from American Type Culture Collection (ATCC). 
Differentiation and Characterization of iPSC-RPE
We differentiated iPSC lines into RPE using a 14-day directed method (see Supplementary Fig. S2).2730 All cell lines showed robust pigmentation by day 30 (P3). Both WT- and LCHADD-RPE formed a monolayer with the characteristic polygonal cobblestone shape and immunofluorescent staining indicated expression of seven common markers found in mature RPE (Fig. 1A). We did not note any clear differences in these markers between cell lines. To examine tight-junctions, WT and LCHADD RPE were grown on Transwell inserts and TEER was determined. In contrast to the study by Polinati et al., TEER was similar between WT- and LCHADD-RPE (Fig. 1B).20 We also used transmission electron microscopy to examine tight junctions; both WT- and LCHADD-RPE appear to have intact tight junctions (Fig. 1C). The common c.1528G>C variant in HADHA is associated with expression of normal amounts of TFPα protein (albeit with impaired LCHAD activity) in cultured human fibroblasts,3 so we anticipated our iPSC-derived RPE would also express TFPα protein. There was similar expression of TFPα, TFPß, and VLCAD, the FAO enzyme immediately upstream of TFP, in both WT- and LCHADD-RPE (Fig. 1D). 
Figure 1.
 
Characterization of iPSC-induced retinal pigment epithelium cells. WT and LCHADD iPSC cell lines were differentiated to RPE and allowed to mature. Cells were stained for common RPE markers and imaged. (A) ZO-1, CRALBP, PMEL, MITF, ATPase, BEST, and RPE65 were not different between cell types. Bright field (BF) images demonstrate both cell lines became confluent octagonal pigmented cells in culture. Scale bar = 100 µm. (B) Transepithelial electrical resistance (TEER) of WT (blue) and LCHADD cells (orange) grown on transwell inserts was similar (data points represent technical replicates, Student t-test > 0.05). (C) TEM shows intact tight junctions (red arrows) in both WT and LCHADD cells. Scale bars = 200 nm. (D) Representative Western blots showing similar TFPα, TFPβ, and VLCAD expression on both WT and LCHADD cell lysates compared to total protein. (E) RPE were incubated with FITC-POS and imaged to determine if cultured RPE could phagocytose POS. (F) Cells were stained for rhodopsin (red) and the overlay demonstrates internalized POS as green and surface bound POS as yellow. Scale bar = 200 µm. (G) The 3-dimensional confocal stack image also indicates internalized POS toward the basal membrane of the LCHADD RPE. Scale bar = 100 µm. Data presented as mean ± SD.
Figure 1.
 
Characterization of iPSC-induced retinal pigment epithelium cells. WT and LCHADD iPSC cell lines were differentiated to RPE and allowed to mature. Cells were stained for common RPE markers and imaged. (A) ZO-1, CRALBP, PMEL, MITF, ATPase, BEST, and RPE65 were not different between cell types. Bright field (BF) images demonstrate both cell lines became confluent octagonal pigmented cells in culture. Scale bar = 100 µm. (B) Transepithelial electrical resistance (TEER) of WT (blue) and LCHADD cells (orange) grown on transwell inserts was similar (data points represent technical replicates, Student t-test > 0.05). (C) TEM shows intact tight junctions (red arrows) in both WT and LCHADD cells. Scale bars = 200 nm. (D) Representative Western blots showing similar TFPα, TFPβ, and VLCAD expression on both WT and LCHADD cell lysates compared to total protein. (E) RPE were incubated with FITC-POS and imaged to determine if cultured RPE could phagocytose POS. (F) Cells were stained for rhodopsin (red) and the overlay demonstrates internalized POS as green and surface bound POS as yellow. Scale bar = 200 µm. (G) The 3-dimensional confocal stack image also indicates internalized POS toward the basal membrane of the LCHADD RPE. Scale bar = 100 µm. Data presented as mean ± SD.
To determine if the iPSC-RPE were functionally similar to human RPE, we measured their ability to phagocytize POS. Mature RPE were fed FITC labeled bovine POS (Fig. 1E). To determine whether the signal from FITC-POS was coming from within the cell or only surface bound, we co-stained for rhodopsin. Rhodopsin staining (red) was overlaid on FITC images to indicate surface bound POS (yellow), whereas the green signal shows internalized POS (Figs. 1F, 1G). Both WT- and LCHADD-RPE phagocytose POS demonstrating that they recapitulate many key characteristics of human RPE cells. 
Impaired Fatty Acid Oxidation in LCHADD-RPE
Previous studies have shown that human RPE express long-chain FAO genes and are capable of oxidizing palmitate in vitro.13,34 To examine FAO, we used the Seashore XFe96 bioanalyzer to examine changes in the OCR with a mitochondrial stress test. WT-RPE treated with 200 mM BSA-palmitate and 1 mM carnitine under low glucose conditions showed a substantial increase in basal respiration when compared to a BSA control (Figs. 2A, 2B). In contrast, the basal OCR was unchanged in LCHADD-RPE in the presence of palmitate (Figs. 2A, 2B). The maximal respiration, which indicates the cells’ ability to rapidly oxidize available substrates after addition of FCCP, was also increased in the WT-RPE but not in the LCHADD-RPE in response to palmitate (Figs. 2A, 2C). Although these cells cannot completely metabolize long-chain fatty acids, they should be able to oxidize medium chain fats which use different chain-specific enzymes for β-oxidation. To test this, we treated WT- and LCHADD-RPE cells with BSA-octanoate (C8). We observed an increase in the maximal respiration in both WT- and LCHADD-RPE with the addition of octanoate (Supplementary Fig. S3). 
Figure 2.
 
WT but not LCHADD RPE oxidize long-chain fatty acids. WT- and LCHADD-RPE were matured on Seahorse plates for 45 days. (A) WT-RPE (blue) increase oxygen consumption rate (OCR) upon incubation with palmitate-BSA (C16) compared to BSA alone but LCHADD-RPE (orange) do not increase OCR in the presence of C16. Two-way repeated measures ANOVA (main effects treatment [Trt], time, and interaction [inter] with post hoc Sidex comparisons for each genotype). (B, C) Mean basal and maximal respiration were increased with C16 for WT- but not for LCHADD-RPE. Unpaired students t-test between BSA and BSA-C16 for each genotype (basal WT P < 0.0001 vs. LCHADD P = 0.12 and maximal WT P < 0.0001 vs. LCHADD P = 0.08). (D) RPE grown on transwells were incubated with BSA-C16 and ketones were measured in the apical chamber. There was a significant increase in ketones in WT cells incubated with C16 (P = 0.0012). In LCHADD cells, ketones increased with C16 incubation but the difference between BSA alone and BSA-C16 was not significant (P = 0.09). (E) LCHADD cells accumulated more triglycerides compared to WT when incubated with BSA-C16. (F) Neutral lipid staining confirmed increased lipid deposition in the LCHADD cells compared to WT. Scale bar = 25 µm. (G) C16, C16:1-OH, and C16:0-OH accumulated in LCHADD cells incubated with BSA-C16 but not in WT cells. Two-way ANOVA main effects genotype, acylcarnitine species, interaction (inter) with post hoc Tukey’s comparison. Data points represent technical replicates, data presented as mean ± SD.**P < 0.01; ***P < 0.001, ****P < 0.0001.
Figure 2.
 
WT but not LCHADD RPE oxidize long-chain fatty acids. WT- and LCHADD-RPE were matured on Seahorse plates for 45 days. (A) WT-RPE (blue) increase oxygen consumption rate (OCR) upon incubation with palmitate-BSA (C16) compared to BSA alone but LCHADD-RPE (orange) do not increase OCR in the presence of C16. Two-way repeated measures ANOVA (main effects treatment [Trt], time, and interaction [inter] with post hoc Sidex comparisons for each genotype). (B, C) Mean basal and maximal respiration were increased with C16 for WT- but not for LCHADD-RPE. Unpaired students t-test between BSA and BSA-C16 for each genotype (basal WT P < 0.0001 vs. LCHADD P = 0.12 and maximal WT P < 0.0001 vs. LCHADD P = 0.08). (D) RPE grown on transwells were incubated with BSA-C16 and ketones were measured in the apical chamber. There was a significant increase in ketones in WT cells incubated with C16 (P = 0.0012). In LCHADD cells, ketones increased with C16 incubation but the difference between BSA alone and BSA-C16 was not significant (P = 0.09). (E) LCHADD cells accumulated more triglycerides compared to WT when incubated with BSA-C16. (F) Neutral lipid staining confirmed increased lipid deposition in the LCHADD cells compared to WT. Scale bar = 25 µm. (G) C16, C16:1-OH, and C16:0-OH accumulated in LCHADD cells incubated with BSA-C16 but not in WT cells. Two-way ANOVA main effects genotype, acylcarnitine species, interaction (inter) with post hoc Tukey’s comparison. Data points represent technical replicates, data presented as mean ± SD.**P < 0.01; ***P < 0.001, ****P < 0.0001.
A second fate for the acetyl-CoA produced from β-oxidation is production of ketone bodies. Because LCHADD is a β-oxidation defect, patients are unable to adequately produce ketones even under stress conditions. Similarly, we expected the LCHADD-RPE would produce less ketones when exposed to palmitate. Ketones are an alternative source of fuel for many tissues and it was recently shown that RPE express HMG-CoA synthase and export ketone bodies apically following exposure to long-chain fatty acids.13 We examined ketone production in mature RPE grown on Transwell supports and treated with BSA-palmitate. Although we observed an increase in apically released β-hydroxybutyrate from both genotypes, the increase was only significant in WT-RPE (P = 0.0012 vs. P = 0.09; Fig. 2D). These results suggest that LCHADD-RPE have impaired long-chain FAO. 
Lipid Accumulation in LCHADD-RPE
Given that LCHADD-RPE have impaired long-chain fatty acid oxidation, we were interested in looking at lipid accumulation. Cell pellets were collected to measure triglyceride concentration. LCHADD-RPE showed increased triglycerides compared to the WT-RPE (Fig. 2E). Both cell lines were treated with either BSA or BSA-palmitate and stained with LipidTox to visualize neutral lipid deposits. In the presence of palmitate, LCHADD-RPE showed an increase in neutral lipids, whereas the WT-RPE did not (Fig. 2F). 
One hallmark of LCHADD in patients is high serum levels of circulating 3-OH-acylcarnitines and previous reports have also correlated high serum 3-OH-acylcarnitine levels with decreased retinal function suggesting these intermediates may be toxic.10,35,36 We, therefore, measured acylcarnitine levels in media of WT- and LCHADD-RPE after treating with BSA or BSA-palmitate. There was an overall increase in acylcarnitines, particularly high levels of palmitoyl (C16) and 3-hydroxypalmitoyl-carnitine (C16-OH), in the LCHADD cells treated with palmitate (Fig. 2G, Supplementary Fig. S4). Interestingly, LCHADD-RPE in culture appear to tolerate high levels of these hydroxy-acylcarnitines for extended periods of time without notable toxicity in the presence of glucose. 
Decreased LCHADD-RPE Viability With H2O2
Several studies have hypothesized impaired FAO may increase oxidative stress.19 We hypothesized that iPSC-derived LCHADD-RPE would have increased susceptibility to oxidative stress when fatty acids were the primary energy substrate. RPE were treated with BSA or BSA-palmitate in low glucose media (2.5 mM), supplemented with 1 mM carnitine for 72 hours, and then challenged for 1 to 2 hours with a H2O2 dose based on the IC50 of WT and LCHADD-RPE. H2O2 containing media was removed, and wells were washed with PBS and replaced with BSA or BSA-palmitate containing media. Viability was then measured 2 to 4 hours later with the Alamar Blue reagent. WT-RPE treated with H2O2 had increased viability when exposed to palmitate, whereas LCHADD cells were less viable under oxidative stress conditions when palmitate was present (Fig. 3A). This indicates that the presence of fat and the absence of glucose increases LCHADD-RPE's susceptibility to oxidative stress. 
Figure 3.
 
LCHADD-RPE are susceptible to oxidative stress. (A) LCHADD-RPE were cultured in low-glucose media with carnitine and BSA or BSA-palmitate (C16). H2O2 was added to culture media at the IC50 for 1 hour, then, media was removed, wells were washed with PBS replaced with fresh culture media, and cell viability was measured approximately 4 hours later. WT cells had increased viability with BSA-C16 but both LCHADD cell lines had significantly reduced viability in the presence of C16 + H2O2. (B) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA) for 5 days as a more physiologically relevant oxidative stress to RPE. Cell viability was lower in both LCHADD cell lines but not different in WT. (C) A similar experiment was performed with BSA and BSA-arachidonic acid (AA) for 13 days. Again, LCHADD cells had reduced viability with BSA-AA but WT cells did not. (D) Quantification of ROS with MitoROS stain after DHA treatment show both LCHADD cell lines had increased ROS compared to WT. Scale bar = 200 µm. (E) Similarly, LCHADD cells had lower total glutathione concentration when treated with DHA. (F) Mature WT- and LCHADD-RPE exposed to low glucose and DHA for 6 days were immunostained with Bodipy C11. There was increased lipid peroxidation in LCHADD-RPE. Merged images show an increased oxidized (green fluorescence)/non-oxidized (red fluorescence) ratio in LCHADD-RPE (orange) than WT-RPE (blue). Scale bar = 100 µm. (G) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA; 200 µM) for 8 days, as well as with N-acetyl-cysteine (NAC; 500 µM) and deuterated DHA (dDHA; 200 µM) and dDHA-NAC. Cell viability was lower in LCHADD-RPE (orange) on DHA but was rescued in the presence of NAC, dDHA, and dDHA-NAC. There was no difference in viability between any of the conditions in WT-RPE (blue). (H) A ferroptosis inhibitor, Ferrostatin-1, improved LCHADD cell viability. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates. Data presented as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure 3.
 
LCHADD-RPE are susceptible to oxidative stress. (A) LCHADD-RPE were cultured in low-glucose media with carnitine and BSA or BSA-palmitate (C16). H2O2 was added to culture media at the IC50 for 1 hour, then, media was removed, wells were washed with PBS replaced with fresh culture media, and cell viability was measured approximately 4 hours later. WT cells had increased viability with BSA-C16 but both LCHADD cell lines had significantly reduced viability in the presence of C16 + H2O2. (B) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA) for 5 days as a more physiologically relevant oxidative stress to RPE. Cell viability was lower in both LCHADD cell lines but not different in WT. (C) A similar experiment was performed with BSA and BSA-arachidonic acid (AA) for 13 days. Again, LCHADD cells had reduced viability with BSA-AA but WT cells did not. (D) Quantification of ROS with MitoROS stain after DHA treatment show both LCHADD cell lines had increased ROS compared to WT. Scale bar = 200 µm. (E) Similarly, LCHADD cells had lower total glutathione concentration when treated with DHA. (F) Mature WT- and LCHADD-RPE exposed to low glucose and DHA for 6 days were immunostained with Bodipy C11. There was increased lipid peroxidation in LCHADD-RPE. Merged images show an increased oxidized (green fluorescence)/non-oxidized (red fluorescence) ratio in LCHADD-RPE (orange) than WT-RPE (blue). Scale bar = 100 µm. (G) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA; 200 µM) for 8 days, as well as with N-acetyl-cysteine (NAC; 500 µM) and deuterated DHA (dDHA; 200 µM) and dDHA-NAC. Cell viability was lower in LCHADD-RPE (orange) on DHA but was rescued in the presence of NAC, dDHA, and dDHA-NAC. There was no difference in viability between any of the conditions in WT-RPE (blue). (H) A ferroptosis inhibitor, Ferrostatin-1, improved LCHADD cell viability. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates. Data presented as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.
Long-Chain Polyunsaturated Fatty Acids Show Increased Toxicity in LCHADD-RPE
The daily process of phagocytosing POS exposes RPE to a high number of polyunsaturated fatty acid species, such as DHA and arachidonic acid.15,37,38 These PUFAs are susceptible to oxidative damage and, in LCHADD cells, may interact with the accumlating hydroxy fatty acids which may promote lipid oxidation. We therefore tested the effects of a long-term exposure to high doses of either DHA or arachidonic acid on the viability of WT- and LCHADD-RPE. After 5 days of DHA exposure, both LCHADD cell lines showed a dramatic decrease in viability when compared to the BSA control, whereas there was no difference in viability in WT cells treated with BSA or DHA (Fig. 3B). Treatment with arachidonic acid started to impact viability exclusively in the LCHADD-RPE after 13 days (Fig. 3C). 
To determine if the presence of DHA increased oxidative stress, we measured mitochondrial reactive oxygen species after DHA treatment. LCHADD-RPE had increased MitoRos staining after DHA treatment compared to WT-RPE (Fig. 3D). We also measured cellular total glutathione concentration and found lower glutathione in LCHADD-RPE exposed to DHA (Fig. 3E) suggesting a depletion of oxidative defense under PUFA exposure. To assess if DHA treatment of LCHADD-RPE increases oxidative damage, we measured lipid peroxidation after DHA treatment using BODIPY C11. The ratio of oxidized/non-oxidized lipids in LCHADD-RPE was significantly higher compared to WT-RPE, suggesting increased lipid peroxidation (Fig. 3F). 
To determine if LCHADD-RPE can be rescued by preventing oxidative damage, LCHADD-RPE cells exposed to DHA were treated with N-acetylcysteine (NAC), a potent antioxidant that replenishes glutathione reserves. Similarly, to determine if LCHADD-RPE can be rescued by providing a nonoxidizable substrate, DHA exposed LCHADD-RPE were treated with 6,6,9,9,12,12,15,15,18,18-d10-docosahexaenoic acid (dDHA), a novel deuterated DHA that cannot be peroxidized.38,39 Cell viability was significantly increased when compared to LCHADD-RPE exposed to DHA alone (Fig. 3G). WT cells had no change in cell viability between any of the treatments. Overall, long-term exposure to PUFA fatty acids under low glucose conditions is associated with increased oxidative stress and reduced viability in LCHADD-RPE but not WT. Viability is rescued by either a potent antioxidant (NAC) or a deuterated version of DHA that escapes lipid peroxidation. 
Lipid peroxidation can cause cell death through ferroptosis, a process of iron mediated lipid peroxidation that leads to cell death.19 To determine if ferroptosis was increased in LCHADD-RPE challenged with DHA, we measured the ability of the ferroptosis inhibitor, ferrostatin-1 (Fer-1) to rescue cell viability in LCHADD cells treated with DHA. Ferrostatin-1 modestly increased cell viability compared to DMSO alone, but this did not reach significance suggesting that the ferroptosis pathway may not be the only cell death pathway involved in DHA-induced RPE death (Fig. 3H). 
Exogenous TFPα Derived From AAV-HADHA Localizes to the Mitochondria and Interacts With Endogenous TFPβ
To test whether a full-length copy of WT HAHDA cDNA could rescue the LCHADD-RPE phenotypes, we transfected LCHADD-RPE with a recombinant adeno-associated virus (rAAV) expression vector containing 3′ 3XFLAG-tagged human HADHA cDNA under the transcriptional control of the CMV enhancer-CAG promoter (Fig. 4A, Supplementary Fig. S5). To assess whether exogenous HADHA would produce a functional TFPα that interacts with endogenous TFPβ, and localizes properly to the mitochondria, transfected LCHADD-RPE were immunostained for FLAG and VLCAD (Figs. 4B, 4C). FLAG was detectable in transfected cells and co-localized with VLCAD, indicating the AAV5-CAG-HADHA-3XFLAG encodes a stable protein that localizes to the mitochondria (Fig. 4C, Supplementary Fig. S5). To ensure that the exogenous TFPα interacts with TFPβ to form the TFP complex, transfected cells were immunoprecipitated with FLAG. After pulling down proteins with the FLAG antibody, we also detected TFPβ (Fig. 4D) confirming that exogenous TFPα–FLAG does interact with TFPβ. These data suggest that the AAV5-CAG-HADHA-3XFLAG encodes for a stable TFPα protein that incorporates into the TFP complex and properly localizes to the mitochondria. 
Figure 4.
 
Exogenous TFP α localizes to the mitochondria and interacts with TFPβ. (A) Schematic representation of the rAAV expression vector containing 3′ 3xFLAG-tagged human HADHA under the transcriptional control of CAG promoter. (B) Cells were fixed and immunostained for FLAG after LCHADD-RPE cells were matured and transduced with AAV-HADHA-3xFLAG. Detection of FLAG (green fluorescence) demonstrates the AAV-HADHA-3XFLAG expresses a stable protein that can be detected using the FLAG antibody. Detection of GFP in LCHADD RPE cells transduced with an AAV-GFP was used as a positive control for transfection method. Scale bar = 200 µm. (C) Transduced LCHADD-RPE cells were stained with VLCAD (red) and FLAG (green) antibodies to determine proper localization to the mitochondria. The yellow merged image shows co-localization of the encoded protein with the mitochondrial FAO enzyme, suggesting proper localization to the mitochondria. Scale bar = 50 µm. (D) Immunoprecipitation of RPE lysates show an interaction of FLAG and endogenous TFPβ, suggesting the transgene encodes for a TFPα-FLAG protein that can interact with endogenous TFPβ.
Figure 4.
 
Exogenous TFP α localizes to the mitochondria and interacts with TFPβ. (A) Schematic representation of the rAAV expression vector containing 3′ 3xFLAG-tagged human HADHA under the transcriptional control of CAG promoter. (B) Cells were fixed and immunostained for FLAG after LCHADD-RPE cells were matured and transduced with AAV-HADHA-3xFLAG. Detection of FLAG (green fluorescence) demonstrates the AAV-HADHA-3XFLAG expresses a stable protein that can be detected using the FLAG antibody. Detection of GFP in LCHADD RPE cells transduced with an AAV-GFP was used as a positive control for transfection method. Scale bar = 200 µm. (C) Transduced LCHADD-RPE cells were stained with VLCAD (red) and FLAG (green) antibodies to determine proper localization to the mitochondria. The yellow merged image shows co-localization of the encoded protein with the mitochondrial FAO enzyme, suggesting proper localization to the mitochondria. Scale bar = 50 µm. (D) Immunoprecipitation of RPE lysates show an interaction of FLAG and endogenous TFPβ, suggesting the transgene encodes for a TFPα-FLAG protein that can interact with endogenous TFPβ.
Expression of AAV-HADHA Improves Fatty Acid Oxidation, Rescues Ketone Production, and Viability in LCHADD-RPE
To determine if adding an exogenous HADHA will restore long-chain FAO, basal OCR was examined in transfected LCHADD-RPE treated with BSA-palmitate using the Seahorse XFe96 bioanalyzer. Similar to WT-RPE, the addition of palmitate to transfected LCHADD-RPE significantly increased the OCR whereas non-transfected LCHADD-RPE had no change in basal OCR (Fig. 5A), indicating that long-chain FAO was restored in transfected RPE. Transfected LCHADD-RPE released significantly more ketones in response to palmitate (Fig. 5B), remained viable after a long-term DHA treatment (Fig. 5C), and had significantly less long-chain acylcarnitines and hydroxyacylcarnitines after being challenged with palmitate (Fig. 5D) when compared to LCHADD cells expressing AAV-GFP or untreated LCHADD cells. Overall, exogenous HADHA delivered with an rAAV rescued the LCHADD-RPE phenotype and highlights the therapeutic potential of gene addition for LCHADD chorioretinopathy. 
Figure 5.
 
AAV-HADHA rescues LCHADD phenotype in RPE. (A) LCHADD-RPE cells transduced with AAV-HADHA-3xFLAG increased oxygen consumption rate (OCR) with BSA-C16 and carnitine. (B) LCHADD cells transduced with AAV-HADHA-3xFLAG had increased ketone production and (C) increased viability with DHA treatment compared to LCHADD cells treated with an AAV-GFP. (D) Treating LCHADD cells with AAV-HADHA-3xFLAG significantly decreased 3-OH-acylcarnitine accumulation compared to untreated cells. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates, data presented as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Figure 5.
 
AAV-HADHA rescues LCHADD phenotype in RPE. (A) LCHADD-RPE cells transduced with AAV-HADHA-3xFLAG increased oxygen consumption rate (OCR) with BSA-C16 and carnitine. (B) LCHADD cells transduced with AAV-HADHA-3xFLAG had increased ketone production and (C) increased viability with DHA treatment compared to LCHADD cells treated with an AAV-GFP. (D) Treating LCHADD cells with AAV-HADHA-3xFLAG significantly decreased 3-OH-acylcarnitine accumulation compared to untreated cells. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates, data presented as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Discussion
In an effort to understand the pathophysiology of LCHADD chorioretinopathy, we established an iPSC-derived RPE cellular model. Mature LCHADD-RPE express RPE markers, have the characteristic monolayer polygonal cobblestone appearance, can form tight junctions, and internalize POSs (see Fig. 1). Additionally, we demonstrated that LCHADD-RPE recapitulate LCHADD markers, such as high levels of hydroxy-acylcarnitines, impaired long-chain FAO, and reduced production of ketones (see Fig. 2). These LCHADD iPSC-derived RPE provide a reproducible in vitro model to study LCHADD chorioretinopathy. 
Healthy RPE are exposed to high levels of oxidative stress and are required to metabolize and recycle PUFAs from POS.40 Oxidation of PUFAs by ROS initiates a lipid peroxidation chain reaction producing many lipid peroxides that healthy RPE can withstand with adequate cellular antioxidants, such as glutathione to quench the ROS as demonstrated by WT-RPE treated with H2O2 and DHA. However, this stress is not well tolerated by LCHADD-RPE that cannot metabolize high concentrations of PUFAs effectively. In the presence of PUFAs, we show that LCHADD-RPE have reduced mitochondrial respiration and accumulation of partially oxidized fatty acids with a 3-hydroxyl group (see Figs. 2A–C, 2G). They also show increased oxidative stress, oxidative damage, and cell death (see Figs. 3B–F). We propose that accumulation of 3-hydroxy fatty acids and 3-hydroxyacylcarnitines observed in LCHADD-RPE increase cellular susceptibility to oxidative stress-induced cell death in highly oxidative environments, potentially explaining the RPE loss observed in patients with LCHADD. 
In the highly oxidative environment of the retina, DHA is susceptible to lipid peroxidation with the creation of products, like carboxyethylpyrrole, which propagate peroxidation and tissue damage but it remains unclear which cellular pathway is activated in LCHADD-RPE exposed to PUFAs.18 Here, we demonstrated that LCHADD-RPE are susceptible to cell death when exposed to PUFAs due to oxidative stress and lipid peroxidation and that a potent antioxidant, NAC, rescues LCHADD-RPE from cell death. Additionally, the presence of dDHA, a modified DHA immune to peroxidation,38,39 also protects LCHADD-RPE from oxidative stress and death (see Fig. 3).38 Our results are encouraging as the presence of NAC or dDHA protects LCHADD-RPE against cell death induced by long-term exposure to PUFAs, such as DHA. 
LCHADD is commonly caused by the presence of a pathogenic variant c.1528G>C in HADHA which causes a change in the catalytic portion of the enzyme reducing its ability to oxidize long-chain hydroxy fatty acids. We hypothesized that gene addition of a WT copy of HADHA could rescue LCHADD-RPE. We transduced mature LCHADD-RPE with AAV-HADHA-3XFLAG and show that it was incorporated into the cells and co-localized with VLCAD in the mitochondria. Transduced cells had restored FAO, reduced hydroxy-acylcarnitines, improved ketone production, and increased viability when exposed to DHA. This suggests gene addition therapy may be a viable potential treatment for chorioretinopathy in patients with LCHADD. 
This study has a few limitations. First, these are patient-derived iPSC-RPE, so specific patient variability is still possible. We have tested two different patient cell lines but additional studies in a variety of iPSC-derived patient lines to account for individual variability are needed. A larger sample size is also needed to confirm these findings. Second, while our studies demonstrate that oxidative stress and lipid peroxidation are involved in cell death, the specific pathway is still not completely elucidated. Further studies using this cell model are needed to understand the mitochondrial dysregulation involved in RPE degeneration leading to chorioretinopathy in patients with LCHADD. Finally, although our gene addition therapy results are promising, this is a preclinical finding, so further translational studies are required to explore retinal gene addition as a viable option for patients living with LCHADD. 
In conclusion, iPSC-derived mature RPE cells carrying the homozygous c.1528G>C common pathogenic variant for LCHADD oxidize less fat, produce less ketones, accumulate 3-hydroxy acylcarnitines, and have reduced viability when exposed to PUFAs. Additionally, LCHADD-RPE have a reduced tolerance to oxidative stress and lipid peroxidation, which can be rescued by potent antioxidants like NAC and dDHA. Furthermore, when these cells express a WT TFPα by gene addition, mature LCHADD-RPE have restored FAO capacity, as documented by increased ketone production, significantly less accumulation of 3-hydroxyacylcarnitines, and no susceptibility to cell death when exposed to DHA, indicating an increased tolerance to oxidative stress. Because RPE is a metabolically active specialized tissue that needs to respond quickly to different stimuli, a microenvironment with high amounts of partially oxidized 3-hydroxy fatty acids reduces the RPE capacity to withstand lipid peroxidation and oxidative stress potentially leading to RPE loss. Eventually, the loss of the RPE leads to loss of the choriocapillaris and large choroidal vessels and ultimately loss of photoreceptors observed in patients with LCHADD. Altering the cell's microenvironment by exposing them to NAC or dDHA, as well as gene addition therapy, prevents cell death and represent promising therapeutic approaches worth investigating. 
Acknowledgments
The authors would like to thank the patients and their families for their kind cooperation. 
Supported by a grant from the Scully-Peterson Foundation and by a grant from the National Eye Institute (1R01EY032889). The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health. 
Author Contributions: Conceptualization: M.B.G., T.D., G.G., and C.O.H. Data curation: T.D., G.E., G.G., and S.B. Formal analysis: T.D., G.E., and M.B.G. Acylcarnitine analysis: D.M.; provided dDHA: M.S.S. Visualization: T.D., G.E., and M.B.G., Writing – original draft: T.D. and G.E. Writing – review and editing: T.D., G.E., G.G., S.B., M.B.G., C.O.H., D.M., M.S.S., and M.E.P. 
Ethics Declaration: Written informed consent was obtained from all participants for use of fibroblasts as part of the Fatty Acid Oxidation Disorders Repository (eIRB#8561). A separate institutional review board (IRB) approval for the use of stored fibroblasts from the Repository for this project was approved by the OHSU IRB (eIRB#9254). All studies adhered to the principles in the Declaration of Helsinki. 
Data Availability: Data will be available upon request to the corresponding author, Dr. Melanie Gillingham at [email protected]
Disclosure: T. DeVine, None; G. Elizondo, None; G. Gaston, None; S.J. Babcock, None; D. Matern, None; M.S. Shchepinov, Retrotrope, Inc. (E) that provided the dDHA; M.E. Pennesi, 4D Molecular Therapeutics (C), Adverum (C), Arrowhead Pharmaceuticals (C), AGTC (C), Aldebraran (C), Ascidian (C), Atsena (C), Astellas (C), BlueRock-Opsis (C), Coave (C), ClarisBio (C), Dompe (C), Editas (C), Edigene (C), Endogena (C), FFB (C), Ingel Therapeutics J-Cyte (C), Janssen (C), KalaTherapeutics (C), Kiora (C), Nacuity Pharmaceuticals (C), Ocugen (C), Ora (C), ProQR (C), Prime Editing (C), PTC Therapeutics (C), PYC Therapeutics (C), Ray Therapeutics (C), Rejuvitas (C), RestoreVision (C), RegenexBio (C), Sparing Vision (C), SpliceBio (C), Spotlight Therapeutics (C), Thea (C), Theranexus (C), AGTC (F), Biogen (F), Editas (F), FFB (F), ProQR (F), Reneuron (F), Data Safety Montitoring Board (DSMB) for Akous (R), Gensight (R), Aldebaran (I), Atsena (I), Endogena (I), EnterX (I), Ingel Therapeutics (I), Kiora (I), Nacuity Pharmaceuticals (I), Ocugen (I), and ZipBio (I); C.O. Harding, Reneo Pharmaceutical (F), Nestle Bioscience (F); M.B. Gillingham, Ultragenyx Pharmaceutical Inc. (S), Vitaflow (S), and Nutricia (S), and Nestle Bioscience (F), Reneo Pharmaceutical (F) 
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Figure 1.
 
Characterization of iPSC-induced retinal pigment epithelium cells. WT and LCHADD iPSC cell lines were differentiated to RPE and allowed to mature. Cells were stained for common RPE markers and imaged. (A) ZO-1, CRALBP, PMEL, MITF, ATPase, BEST, and RPE65 were not different between cell types. Bright field (BF) images demonstrate both cell lines became confluent octagonal pigmented cells in culture. Scale bar = 100 µm. (B) Transepithelial electrical resistance (TEER) of WT (blue) and LCHADD cells (orange) grown on transwell inserts was similar (data points represent technical replicates, Student t-test > 0.05). (C) TEM shows intact tight junctions (red arrows) in both WT and LCHADD cells. Scale bars = 200 nm. (D) Representative Western blots showing similar TFPα, TFPβ, and VLCAD expression on both WT and LCHADD cell lysates compared to total protein. (E) RPE were incubated with FITC-POS and imaged to determine if cultured RPE could phagocytose POS. (F) Cells were stained for rhodopsin (red) and the overlay demonstrates internalized POS as green and surface bound POS as yellow. Scale bar = 200 µm. (G) The 3-dimensional confocal stack image also indicates internalized POS toward the basal membrane of the LCHADD RPE. Scale bar = 100 µm. Data presented as mean ± SD.
Figure 1.
 
Characterization of iPSC-induced retinal pigment epithelium cells. WT and LCHADD iPSC cell lines were differentiated to RPE and allowed to mature. Cells were stained for common RPE markers and imaged. (A) ZO-1, CRALBP, PMEL, MITF, ATPase, BEST, and RPE65 were not different between cell types. Bright field (BF) images demonstrate both cell lines became confluent octagonal pigmented cells in culture. Scale bar = 100 µm. (B) Transepithelial electrical resistance (TEER) of WT (blue) and LCHADD cells (orange) grown on transwell inserts was similar (data points represent technical replicates, Student t-test > 0.05). (C) TEM shows intact tight junctions (red arrows) in both WT and LCHADD cells. Scale bars = 200 nm. (D) Representative Western blots showing similar TFPα, TFPβ, and VLCAD expression on both WT and LCHADD cell lysates compared to total protein. (E) RPE were incubated with FITC-POS and imaged to determine if cultured RPE could phagocytose POS. (F) Cells were stained for rhodopsin (red) and the overlay demonstrates internalized POS as green and surface bound POS as yellow. Scale bar = 200 µm. (G) The 3-dimensional confocal stack image also indicates internalized POS toward the basal membrane of the LCHADD RPE. Scale bar = 100 µm. Data presented as mean ± SD.
Figure 2.
 
WT but not LCHADD RPE oxidize long-chain fatty acids. WT- and LCHADD-RPE were matured on Seahorse plates for 45 days. (A) WT-RPE (blue) increase oxygen consumption rate (OCR) upon incubation with palmitate-BSA (C16) compared to BSA alone but LCHADD-RPE (orange) do not increase OCR in the presence of C16. Two-way repeated measures ANOVA (main effects treatment [Trt], time, and interaction [inter] with post hoc Sidex comparisons for each genotype). (B, C) Mean basal and maximal respiration were increased with C16 for WT- but not for LCHADD-RPE. Unpaired students t-test between BSA and BSA-C16 for each genotype (basal WT P < 0.0001 vs. LCHADD P = 0.12 and maximal WT P < 0.0001 vs. LCHADD P = 0.08). (D) RPE grown on transwells were incubated with BSA-C16 and ketones were measured in the apical chamber. There was a significant increase in ketones in WT cells incubated with C16 (P = 0.0012). In LCHADD cells, ketones increased with C16 incubation but the difference between BSA alone and BSA-C16 was not significant (P = 0.09). (E) LCHADD cells accumulated more triglycerides compared to WT when incubated with BSA-C16. (F) Neutral lipid staining confirmed increased lipid deposition in the LCHADD cells compared to WT. Scale bar = 25 µm. (G) C16, C16:1-OH, and C16:0-OH accumulated in LCHADD cells incubated with BSA-C16 but not in WT cells. Two-way ANOVA main effects genotype, acylcarnitine species, interaction (inter) with post hoc Tukey’s comparison. Data points represent technical replicates, data presented as mean ± SD.**P < 0.01; ***P < 0.001, ****P < 0.0001.
Figure 2.
 
WT but not LCHADD RPE oxidize long-chain fatty acids. WT- and LCHADD-RPE were matured on Seahorse plates for 45 days. (A) WT-RPE (blue) increase oxygen consumption rate (OCR) upon incubation with palmitate-BSA (C16) compared to BSA alone but LCHADD-RPE (orange) do not increase OCR in the presence of C16. Two-way repeated measures ANOVA (main effects treatment [Trt], time, and interaction [inter] with post hoc Sidex comparisons for each genotype). (B, C) Mean basal and maximal respiration were increased with C16 for WT- but not for LCHADD-RPE. Unpaired students t-test between BSA and BSA-C16 for each genotype (basal WT P < 0.0001 vs. LCHADD P = 0.12 and maximal WT P < 0.0001 vs. LCHADD P = 0.08). (D) RPE grown on transwells were incubated with BSA-C16 and ketones were measured in the apical chamber. There was a significant increase in ketones in WT cells incubated with C16 (P = 0.0012). In LCHADD cells, ketones increased with C16 incubation but the difference between BSA alone and BSA-C16 was not significant (P = 0.09). (E) LCHADD cells accumulated more triglycerides compared to WT when incubated with BSA-C16. (F) Neutral lipid staining confirmed increased lipid deposition in the LCHADD cells compared to WT. Scale bar = 25 µm. (G) C16, C16:1-OH, and C16:0-OH accumulated in LCHADD cells incubated with BSA-C16 but not in WT cells. Two-way ANOVA main effects genotype, acylcarnitine species, interaction (inter) with post hoc Tukey’s comparison. Data points represent technical replicates, data presented as mean ± SD.**P < 0.01; ***P < 0.001, ****P < 0.0001.
Figure 3.
 
LCHADD-RPE are susceptible to oxidative stress. (A) LCHADD-RPE were cultured in low-glucose media with carnitine and BSA or BSA-palmitate (C16). H2O2 was added to culture media at the IC50 for 1 hour, then, media was removed, wells were washed with PBS replaced with fresh culture media, and cell viability was measured approximately 4 hours later. WT cells had increased viability with BSA-C16 but both LCHADD cell lines had significantly reduced viability in the presence of C16 + H2O2. (B) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA) for 5 days as a more physiologically relevant oxidative stress to RPE. Cell viability was lower in both LCHADD cell lines but not different in WT. (C) A similar experiment was performed with BSA and BSA-arachidonic acid (AA) for 13 days. Again, LCHADD cells had reduced viability with BSA-AA but WT cells did not. (D) Quantification of ROS with MitoROS stain after DHA treatment show both LCHADD cell lines had increased ROS compared to WT. Scale bar = 200 µm. (E) Similarly, LCHADD cells had lower total glutathione concentration when treated with DHA. (F) Mature WT- and LCHADD-RPE exposed to low glucose and DHA for 6 days were immunostained with Bodipy C11. There was increased lipid peroxidation in LCHADD-RPE. Merged images show an increased oxidized (green fluorescence)/non-oxidized (red fluorescence) ratio in LCHADD-RPE (orange) than WT-RPE (blue). Scale bar = 100 µm. (G) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA; 200 µM) for 8 days, as well as with N-acetyl-cysteine (NAC; 500 µM) and deuterated DHA (dDHA; 200 µM) and dDHA-NAC. Cell viability was lower in LCHADD-RPE (orange) on DHA but was rescued in the presence of NAC, dDHA, and dDHA-NAC. There was no difference in viability between any of the conditions in WT-RPE (blue). (H) A ferroptosis inhibitor, Ferrostatin-1, improved LCHADD cell viability. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates. Data presented as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure 3.
 
LCHADD-RPE are susceptible to oxidative stress. (A) LCHADD-RPE were cultured in low-glucose media with carnitine and BSA or BSA-palmitate (C16). H2O2 was added to culture media at the IC50 for 1 hour, then, media was removed, wells were washed with PBS replaced with fresh culture media, and cell viability was measured approximately 4 hours later. WT cells had increased viability with BSA-C16 but both LCHADD cell lines had significantly reduced viability in the presence of C16 + H2O2. (B) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA) for 5 days as a more physiologically relevant oxidative stress to RPE. Cell viability was lower in both LCHADD cell lines but not different in WT. (C) A similar experiment was performed with BSA and BSA-arachidonic acid (AA) for 13 days. Again, LCHADD cells had reduced viability with BSA-AA but WT cells did not. (D) Quantification of ROS with MitoROS stain after DHA treatment show both LCHADD cell lines had increased ROS compared to WT. Scale bar = 200 µm. (E) Similarly, LCHADD cells had lower total glutathione concentration when treated with DHA. (F) Mature WT- and LCHADD-RPE exposed to low glucose and DHA for 6 days were immunostained with Bodipy C11. There was increased lipid peroxidation in LCHADD-RPE. Merged images show an increased oxidized (green fluorescence)/non-oxidized (red fluorescence) ratio in LCHADD-RPE (orange) than WT-RPE (blue). Scale bar = 100 µm. (G) RPE were cultured in low-glucose media with carnitine and BSA or BSA-docosahexaenoic acid (DHA; 200 µM) for 8 days, as well as with N-acetyl-cysteine (NAC; 500 µM) and deuterated DHA (dDHA; 200 µM) and dDHA-NAC. Cell viability was lower in LCHADD-RPE (orange) on DHA but was rescued in the presence of NAC, dDHA, and dDHA-NAC. There was no difference in viability between any of the conditions in WT-RPE (blue). (H) A ferroptosis inhibitor, Ferrostatin-1, improved LCHADD cell viability. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates. Data presented as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.
Figure 4.
 
Exogenous TFP α localizes to the mitochondria and interacts with TFPβ. (A) Schematic representation of the rAAV expression vector containing 3′ 3xFLAG-tagged human HADHA under the transcriptional control of CAG promoter. (B) Cells were fixed and immunostained for FLAG after LCHADD-RPE cells were matured and transduced with AAV-HADHA-3xFLAG. Detection of FLAG (green fluorescence) demonstrates the AAV-HADHA-3XFLAG expresses a stable protein that can be detected using the FLAG antibody. Detection of GFP in LCHADD RPE cells transduced with an AAV-GFP was used as a positive control for transfection method. Scale bar = 200 µm. (C) Transduced LCHADD-RPE cells were stained with VLCAD (red) and FLAG (green) antibodies to determine proper localization to the mitochondria. The yellow merged image shows co-localization of the encoded protein with the mitochondrial FAO enzyme, suggesting proper localization to the mitochondria. Scale bar = 50 µm. (D) Immunoprecipitation of RPE lysates show an interaction of FLAG and endogenous TFPβ, suggesting the transgene encodes for a TFPα-FLAG protein that can interact with endogenous TFPβ.
Figure 4.
 
Exogenous TFP α localizes to the mitochondria and interacts with TFPβ. (A) Schematic representation of the rAAV expression vector containing 3′ 3xFLAG-tagged human HADHA under the transcriptional control of CAG promoter. (B) Cells were fixed and immunostained for FLAG after LCHADD-RPE cells were matured and transduced with AAV-HADHA-3xFLAG. Detection of FLAG (green fluorescence) demonstrates the AAV-HADHA-3XFLAG expresses a stable protein that can be detected using the FLAG antibody. Detection of GFP in LCHADD RPE cells transduced with an AAV-GFP was used as a positive control for transfection method. Scale bar = 200 µm. (C) Transduced LCHADD-RPE cells were stained with VLCAD (red) and FLAG (green) antibodies to determine proper localization to the mitochondria. The yellow merged image shows co-localization of the encoded protein with the mitochondrial FAO enzyme, suggesting proper localization to the mitochondria. Scale bar = 50 µm. (D) Immunoprecipitation of RPE lysates show an interaction of FLAG and endogenous TFPβ, suggesting the transgene encodes for a TFPα-FLAG protein that can interact with endogenous TFPβ.
Figure 5.
 
AAV-HADHA rescues LCHADD phenotype in RPE. (A) LCHADD-RPE cells transduced with AAV-HADHA-3xFLAG increased oxygen consumption rate (OCR) with BSA-C16 and carnitine. (B) LCHADD cells transduced with AAV-HADHA-3xFLAG had increased ketone production and (C) increased viability with DHA treatment compared to LCHADD cells treated with an AAV-GFP. (D) Treating LCHADD cells with AAV-HADHA-3xFLAG significantly decreased 3-OH-acylcarnitine accumulation compared to untreated cells. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates, data presented as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Figure 5.
 
AAV-HADHA rescues LCHADD phenotype in RPE. (A) LCHADD-RPE cells transduced with AAV-HADHA-3xFLAG increased oxygen consumption rate (OCR) with BSA-C16 and carnitine. (B) LCHADD cells transduced with AAV-HADHA-3xFLAG had increased ketone production and (C) increased viability with DHA treatment compared to LCHADD cells treated with an AAV-GFP. (D) Treating LCHADD cells with AAV-HADHA-3xFLAG significantly decreased 3-OH-acylcarnitine accumulation compared to untreated cells. Unpaired Student t-test and 2-way ANOVA with Tukey's multiple comparisons test were used for statistical analysis. Data points represent technical replicates, data presented as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001.
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