HIF2α activation and mitochondrial deficit due to iron chelation cause retinal atrophy
2023; Springer Nature; Volume: 15; Issue: 2 Linguagem: Inglês
10.15252/emmm.202216525
ISSN1757-4684
AutoresYang Kong, Pei‐Kang Liu, Yao Li, Nicholas D. Nolan, Peter M. J. Quinn, Chun‐Wei Hsu, Laura A. Jenny, Jin Zhao, Xuan Cui, Ya‐Ju Chang, Katherine J. Wert, Janet R. Sparrow, Nan‐Kai Wang, Stephen H. Tsang,
Tópico(s)Retinal Development and Disorders
ResumoArticle16 January 2023Open Access Source DataTransparent process HIF2α activation and mitochondrial deficit due to iron chelation cause retinal atrophy Yang Kong Yang Kong orcid.org/0000-0003-3925-5572 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Pei-Kang Liu Pei-Kang Liu orcid.org/0000-0002-6417-4363 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Yao Li Yao Li Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Resources, Investigation, Methodology Search for more papers by this author Nicholas D Nolan Nicholas D Nolan orcid.org/0000-0002-9236-8309 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Department of Biomedical Engineering, The Fu Foundation School of Engineering and Applied Science, Columbia University, New York, NY, USA Contribution: Formal analysis, Investigation, Visualization, Writing - review & editing Search for more papers by this author Peter M J Quinn Peter M J Quinn orcid.org/0000-0001-9940-4264 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Data curation, Investigation, Visualization, Methodology Search for more papers by this author Chun-Wei Hsu Chun-Wei Hsu Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Validation, Investigation, Visualization, Methodology Search for more papers by this author Laura A Jenny Laura A Jenny orcid.org/0000-0001-8959-7074 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Formal analysis, Visualization, Project administration, Writing - review & editing Search for more papers by this author Jin Zhao Jin Zhao orcid.org/0000-0002-1934-6445 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Investigation, Methodology, Project administration, Writing - review & editing Search for more papers by this author Xuan Cui Xuan Cui Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Conceptualization, Investigation, Methodology Search for more papers by this author Ya-Ju Chang Ya-Ju Chang Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Data curation, Visualization, Methodology Search for more papers by this author Katherine J Wert Katherine J Wert orcid.org/0000-0002-8430-2916 Departments of Ophthalmology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA The Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Janet R Sparrow Janet R Sparrow Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Resources, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Nan-Kai Wang Nan-Kai Wang orcid.org/0000-0002-6277-9879 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Writing - review & editing Search for more papers by this author Stephen H Tsang Corresponding Author Stephen H Tsang [email protected] orcid.org/0000-0001-9082-2427 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Jonas Children's Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Columbia Stem Cell Initiative, Pathology and Cell Biology, Institute of Human Nutrition, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing - review & editing Search for more papers by this author Yang Kong Yang Kong orcid.org/0000-0003-3925-5572 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Pei-Kang Liu Pei-Kang Liu orcid.org/0000-0002-6417-4363 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan Contribution: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Yao Li Yao Li Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Resources, Investigation, Methodology Search for more papers by this author Nicholas D Nolan Nicholas D Nolan orcid.org/0000-0002-9236-8309 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Department of Biomedical Engineering, The Fu Foundation School of Engineering and Applied Science, Columbia University, New York, NY, USA Contribution: Formal analysis, Investigation, Visualization, Writing - review & editing Search for more papers by this author Peter M J Quinn Peter M J Quinn orcid.org/0000-0001-9940-4264 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Data curation, Investigation, Visualization, Methodology Search for more papers by this author Chun-Wei Hsu Chun-Wei Hsu Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Validation, Investigation, Visualization, Methodology Search for more papers by this author Laura A Jenny Laura A Jenny orcid.org/0000-0001-8959-7074 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Formal analysis, Visualization, Project administration, Writing - review & editing Search for more papers by this author Jin Zhao Jin Zhao orcid.org/0000-0002-1934-6445 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Investigation, Methodology, Project administration, Writing - review & editing Search for more papers by this author Xuan Cui Xuan Cui Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Conceptualization, Investigation, Methodology Search for more papers by this author Ya-Ju Chang Ya-Ju Chang Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Data curation, Visualization, Methodology Search for more papers by this author Katherine J Wert Katherine J Wert orcid.org/0000-0002-8430-2916 Departments of Ophthalmology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA The Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Contribution: Conceptualization, Writing - review & editing Search for more papers by this author Janet R Sparrow Janet R Sparrow Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Resources, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Nan-Kai Wang Nan-Kai Wang orcid.org/0000-0002-6277-9879 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Writing - review & editing Search for more papers by this author Stephen H Tsang Corresponding Author Stephen H Tsang [email protected] orcid.org/0000-0001-9082-2427 Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Jonas Children's Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Columbia Stem Cell Initiative, Pathology and Cell Biology, Institute of Human Nutrition, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing - review & editing Search for more papers by this author Author Information Yang Kong1,†, Pei-Kang Liu1,2,3,4,†, Yao Li1,†, Nicholas D Nolan1,5, Peter M J Quinn1, Chun-Wei Hsu1, Laura A Jenny1, Jin Zhao1, Xuan Cui1, Ya-Ju Chang1, Katherine J Wert6,7, Janet R Sparrow1, Nan-Kai Wang1 and Stephen H Tsang *,1,8 1Department of Ophthalmology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA 2Department of Ophthalmology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 3School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan 5Department of Biomedical Engineering, The Fu Foundation School of Engineering and Applied Science, Columbia University, New York, NY, USA 6Departments of Ophthalmology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 7The Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 8Jonas Children's Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Columbia Stem Cell Initiative, Pathology and Cell Biology, Institute of Human Nutrition, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA † These authors contributed equally to this work *Corresponding author. Tel: +212-342-1186; E-mail: [email protected] EMBO Mol Med (2023)15:e16525https://doi.org/10.15252/emmm.202216525 See also: B Rosin & J.-A. Sahel (February 2023) PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Iron accumulation causes cell death and disrupts tissue functions, which necessitates chelation therapy to reduce iron overload. However, clinical utilization of deferoxamine (DFO), an iron chelator, has been documented to give rise to systemic adverse effects, including ocular toxicity. This study provided the pathogenic and molecular basis for DFO-related retinopathy and identified retinal pigment epithelium (RPE) as the target tissue in DFO-related retinopathy. Our modeling demonstrated the susceptibility of RPE to DFO compared with the neuroretina. Intriguingly, we established upregulation of hypoxia inducible factor (HIF) 2α and mitochondrial deficit as the most prominent pathogenesis underlying the RPE atrophy. Moreover, suppressing hyperactivity of HIF2α and preserving mitochondrial dysfunction by α-ketoglutarate (AKG) protects the RPE against lesions both in vitro and in vivo. This supported our observation that AKG supplementation alleviates visual impairment in a patient undergoing DFO-chelation therapy. Overall, our study established a significant role of iron deficiency in initiating DFO-related RPE atrophy. Inhibiting HIF2α and rescuing mitochondrial function by AKG protect RPE cells and can potentially ameliorate patients' visual function. Synopsis Deferoxamine (DFO) stabilizes HIF2α and disrupts mitochondrial oxidative phosphorylation, leading to RPE atrophy. Inhibiting HIF2α and preserving mitochondrial oxidative phosphorylation by α-ketoglutarate (AKG) mitigate RPE cell death and ameliorate visual impairment in the clinic. DFO primarily acts on RPE and disrupts its iron homeostasis, causing atrophic lesions. DFO upregulates HIF2α and undermines mitochondrial oxidative phosphorylation in the RPE, accounting for its susceptibility to iron depletion. RPE intolerance to HIF2α-induced anaerobic glycolysis contributes to susceptibility to DFO toxicity. RPE survival is sustained by mitochondrial oxidative phosphorylation. The glycolytic photoreceptor is initially spared from the DFO-enhanced HIF levels. AKG, an intermediary metabolite of the Krebs cycle, destabilizes HIF2α and preserves mitochondrial respiration capacity, which protect RPE from damage and mitigates visual impairment in the clinic. Introduction Iron is essential in various physiological processes across species, including oxygen transport, energy production and enzymatic catalysis. However, mounting evidence has revealed the consequences of iron excess in disrupting cellular homeostasis and causing cell death (Gozzelino & Arosio, 2016; Eid et al, 2017). Of note, free iron contributes to the production of reactive oxygen species (ROS) that trigger programmed cell death, as exemplified by ferroptosis, a newly-identified form of cell death (Dixon et al, 2012). In the clinic, iron overload, as seen in transfusion-dependent thalassemia patients, results in malfunction across tissues and organs (Kohgo et al, 2008; Shah et al, 2019). Chelation therapy is therefore, indispensable to reducing excess iron and minimizing its systemic toxicity. The advent of deferoxamine (DFO), an iron chelator, significantly improved the life expectancy of transfusion-dependent thalassemia patients as it minimizes systemic complications linked to iron overload (Cohen et al, 1984; Brittenham et al, 1994; Borgna-Pignatti et al, 2004; Poggiali et al, 2012). However, adverse effects of iron chelation emerged in patients with a history of taking DFO (Brittenham, 2011), which prompted a critical need to understand the delicate iron balance and its governing mechanisms in maintaining cell vitality for the clinical management of DFO toxicity. Iron loss by chelation therapy mainly affects iron-dependent signaling cascades, including mitochondrial oxygen consumption and hypoxia. Coupling oxygen consumption with electron transport is a major function of mitochondria for energy production, in which iron plays an indispensable role. Iron deficiency was reported to be linked to ROS production and profound disruption in mitochondrial respiration (Walter et al, 2002; Fujimaki et al, 2019). Moreover, a defective respiratory chain creates a hypoxic condition and induces activation of hypoxia inducible factor (HIF) α. HIFs are heterodimeric transcription factors that are mainly responsible for oxygen-dependent reactions inside the cell (Wang et al, 1995). The stability and transactivation of HIFα rely on prolyl hydroxylase domain (PHD) protein-mediated hydroxylation, in which Fe2+ and α-ketoglutarate (AKG) function as key co-factors. Supplementing Fe2+ or AKG was reported to suppress HIFα in vitro (Kaelin, 2005; Kaelin & Ratcliffe, 2008). As a master regulator of oxygen homeostasis and aerobic glycolysis, the HIFα system is implicated in multiple biological processes, including angiogenesis, extracellular matrix dynamics and cell survival. Abnormal HIFα due to genetic defects or extracellular cues contributes to congenital defects, inflammation, cardiovascular malfunctions and oncogenesis (Bertout et al, 2008; Majmundar et al, 2010). The HIFα family consists of three major isoforms in mammals: HIF1α and its paralog HIF2α, which overlap in structure, and HIF3α (Semenza, 2012). Unlike the ubiquitous expression of HIF1α, HIF2α is exclusively expressed by specific tissues, such as vascular endothelium, liver parenchyma, kidney epithelium, cornea, thymus, and cerebellar Purkinje cells (Talks et al, 2000). Despite the structural resemblance and functional redundancy between HIF1α and HIF2α, nuanced distinctions in transcriptional regulation have been noted (Ginouves et al, 2008; Mastrogiannaki et al, 2009; Keith et al, 2011; Downes et al, 2018). Understanding the differential impact of HIF1α and HIF2α in a cell-specific manner in both healthy and diseased conditions is necessary to study their contribution to disease initiation and progression and explore their therapeutic potential. Ophthalmic toxicity from DFO was first reported among patients who presented with neurosensory impairment (Davies et al, 1983; Olivieri et al, 1986; Rahi et al, 1986; Baath et al, 2008). Of interest, iron deficiency in the eye significantly undermines metabolic homeostasis of retinal pigment epithelial (RPE) cells (Kanow et al, 2017). Despite histological anomalies in the RPE, detailed pathological features of DFO-related retinopathy and its molecular basis are yet to be defined. We, therefore, sought to investigate the influence of iron deficiency in retinal cells and explore a therapeutic solution to DFO-related retinopathy. By inducing DFO toxicity in mice and cultured RPE derived from human induced pluripotent stem cells (iPSCs), we investigated the mechanism underpinning DFO-related retinopathy. Our clinical and experimental characterization provides new evidence that the RPE is a primary target for DFO toxicity, which raises ROS levels and disturbs mitochondrial respiration. Additionally, we noted stabilization of HIF2α rather than HIF1α in response to DFO toxicity in RPE, which transcriptionally upregulates different clusters of genes pertaining to cell viability, glycolysis, and iron transport. Strikingly, AKG suppresses the hyperactivity of HIF2α and modifies metabolic anomalies associated with DFO toxicity in the RPE. Taken together, this study demonstrates the pathological consequences of iron depletion in RPE caused by DFO. It establishes a profound impact of mitochondrial dysfunction and upregulated HIF2α on RPE atrophy. Destabilizing HIF2α and preserving mitochondrial capacity by AKG prevents RPE cell death and can alleviate patient's visual decline due to DFO intake. Results Ophthalmic phenotyping of patients undergoing DFO treatment Four patients of β-thalassemia intermedia with a history of blood transfusion were subject to iron chelation by DFO for at least 16 years (Table 1). These patients visited the clinic due to visual impairment. Unlike typical signs of iron deposition in the eye, such as corneal iron lines, lens changes, etc., color fundoscopy showed a spectrum of distinctive pathologies, including mottling (Fig 1A), lack of intraretinal pigmentation (Fig 1A and B), diffuse depigmentation (Fig 1A–C), multiple atrophic patches of RPE in the macula and peripapillary areas (Fig 1C and D), as well as choroidal sclerotic vessels (Fig 1D). All these pathological abnormalities were evidence of predominant outer retina/RPE damages and prompted us to determine RPE pathology in initiating and progressing DFO-related degenerative retinopathy. Table 1. Summary of patients' clinical profiles. Case Number Age Gender BCVA (OD/OS) at first visit Duration of chelation therapy Diagnosis Intake of AKG I 53 Male 20/80; 20/80 33 years β-thalassemia intermedia No II 26 Male 20/30; 20/630 17 years β-thalassemia intermedia No III 53 Male CF; 20/630 17 years β-thalassemia intermedia No IV 64 Male 20/150; HM 16 years β-thalassemia intermedia Yes BCVA, best corrected visual acuity; CF, counting fingers; HM, hand motion. Figure 1. Ophthalmic examinations of chelation-dependent thalassemia patients show degenerative changes and functional decline A–D. Color fundus photographs and SW-AF examinations on four patients of β-thalassemia subject to chelation therapy by DFO for at least 16 years. Fundus phenotypes, including RPE mottling and depigmentation in the macula (Case I; Gelman et al, 2014), choroidal sclerosis in the perimacular areas (Case II), peripapillary (Cases III and IV), and subretinal pigmentation (Case IV) were evaluated. RPE lesions including concentric distribution of stippled hyper-autofluorescence at the macula (Case I), large areas of hypo-autofluorescent regions at parapapillary or perimacular area (Cases II and III), and extensive RPE loss in both eyes (Case IV) were observed by SW-AF. E–H. ffERG test on the four patients to analyze the light- and dark-adapted vision. I. ffERG profiling of a healthy individual was obtained as the reference. Data information: In E–I, Y-Axis: microvolts; X-Axis: milliseconds. Download figure Download PowerPoint Short-wavelength fundus autofluorescence (SW-AF) imaging was performed to assess potential RPE malfunction due to its capability of capturing the autofluorescence of bisretinoid, which constitutes lipofuscin deposits inside RPE cells. Normal SW-AF shows homogeneous autofluorescence of RPE with a gradual decline in intensity toward the foveola due to high lutein pigment in the foveal region. Foci of hyper- and hypo-autofluorescence, major signs of morbid or atrophic RPE, respectively (Sparrow et al, 2012; Pole & Ameri, 2021), can be seen in SW-AF images of the patients with DFO retinopathy. Case I showed stippled hyper-autofluorescence mainly in the macular region without obvious hypo-autofluorescence, indicating an RPE injury at the early stage of DFO retinopathy (Fig 1A), whereas Cases II, III and IV displayed multiple areas of RPE loss as indicated by diffuse hypo-autofluorescence (Fig 1B–D). The lesions in the neuroretina were further validated by spectral domain optical coherence tomography (SD-OCT). Case I exhibited a fragmented ellipsoid zone (EZ) linked to an early stage of DFO-related retinopathy, granular hyper-reflective deposits localized to the RPE layer, and thinning of the outer retina (Fig EV1A and B). Case II presented with progressive thinning of the outer nuclear layer and subsidence of the outer plexiform layer with indistinguishable EZ band and external limiting membrane (Fig EV1C and D). Additionally, increased transmission of signals into the choroidal and scleral layers due to extensive RPE atrophy became pronounced in Cases II–IV, which are related to the later stage of degenerative retinopathy (Fig EV1C–H). The image profiling of the four patients, from Case I to IV, delineated the progression of DFO-related retinopathy that is characterized by initial pathology of RPE prior to the secondary photoreceptor damage. Visual function assessment by full-field electroretinography (ffERG) further validated our assessment of disease progression of DFO retinopathy, a typical rod-cone dysfunction involved in a majority of RPE dystrophies, in the four individual patients. The ffERG responses of Case I appeared normal due to an early stage of disease (Fig 1E), whereas a gradual decline in both dark- and light-adapted responses can be seen in the remaining three patients with advanced DFO retinopathy in comparison with a healthy individual (Fig 1F–I). Closer inspection indicated a minor reduction in the amplitudes of dark-adapted rod responses and maximal responses in both eyes of Case II (Fig 1F). Significantly reduced amplitudes of the same responses were noted in both eyes of Cases III and IV (Fig 1G and H). The light-adapted single-flash responses and 30 Hz flicker showed a gradual deterioration from Case II to IV as decreased amplitude and delayed implicit time became more and more pronounced (Fig 1F–H), which suggested a great severity and extensive secondary photoreceptor degeneration. The ffERG measurement corroborated the disease severity of the four patients assessed by the aforementioned retinal imaging. Remarkably, Case IV had a history of taking AKG (2 g/day) as a supplement for 18 months. The patient reported slight enhancement in visual function since the start of AKG supplementation. Dilated fundus examination and multimodal imaging showed no distinguishable progression compared with his earlier fundus examinations shown in Fig 1D. In a retrospective comparison before and after AKG intake, ffERG revealed pan-retinal functional improvement in both light-adapted single-flash cone response (OD: 17–22 μV; OS: 5–19 μV) and 30 Hz flicker response (OD: 8–9 μV; OS: 5–9 μV) after continuous AKG supplementation (Fig EV1I). Despite RPE as the primary target for DFO, this suggests that the secondary photoreceptor malfunction or degeneration might be halted by AKG supplementation. Figure 2. RPE is prone to DFO's toxic effect in vivo as a cause of degenerative retinopathy A–C. Adult C57BL/6J mice (four months old) were intraperitoneally injected with DFO at 100 mg/kg three times a week for three months in a row. SW-AF images were acquired longitudinally at one, two and three months post injection. D, E. SD-OCT was performed on seven-month-old mice subject to six months of DFO injection. Age-matched mice without DFO injection were included as the control. Rectangle indicates outer retina/RPE region. Yellow dashed line indicates the RPE layer. ONL: outer nuclear layer; RPE: retinal pigment epithelium. F, G. ERG was carried out on the mice injected with DFO for six months. Photoreceptor scotopic response was measured under the condition of −3 log cd·s/m2; the mixed response was measured under the condition of 0.417 log cd·s/m2; the photopic response was measured under the condition of 1.48 log cd·s/m2. The statistics are analyzed by unpaired Student's t-test. The results are presented as mean ± S.E.M., n = 4 mice for each group. H. RPE light response was determined by exposing mouse eyes to a series of flash intensities. Age-matched untreated mice were included as the controls. The statistics under the same flash intensity are analyzed by unpaired Student's t-test. The results are presented as mean ± S.E.M., n = 4 mice for each group. *P < 0.05. I, J. Flat mount RPE was harvested from the mice treated with DFO for five months and stained by TUNEL to detect cell death. Hoechst was used for nuclear staining. Untreated mice of similar age were used as the healthy controls. Flat mount images were captured by 10× confocal microscopy and stitched together. Scale bar: 1 mm. K–N. The RPE was dissected from the mice injected with DFO for three and nine months, respectively, and stained with phalloidin. Age-matched untreated mice were included as the controls. The images were taken at 40× magnification. Scale bar: 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The chelation-dependent thalassemia patients show retinal degeneration and functional improvement by taking AKG A–H. SD-OCT of the four thalassemia patients with a history of taking DFO showed interruption of the ellipsoid zone (Case I; Gelman et al, 2014), decreased photoreceptor nuclear layer, and thinning of the retinal layers (Cases II–IV). Focal thickening and bumps of RPE were also noted in Case I. Granular hyper-reflective deposits within the RPE (yellow arrows) can be seen in all four cases. An intraretinal degenerative cyst (Case IV) and multiple areas of choroidal hyper-transmission were also noted in Cases II–IV. I. The ffERG examination on Case IV with continuous supplementation of AKG for 18 months (2 g/day). Repeated ffERG examination (lower panel) on both eyes showed increased amplitudes in light-adapted single-flash cone and 30 Hz flicker responses compared with the amplitude prior to taking AKG (upper panel). The exact numbers of the peak value are indicated inside the panel boxes. Y-Axis: microvolts; X-Axis: milliseconds. Download figure Download PowerPoint Mouse modeling and in vitro characterization of DFO toxicity in RPE To better understand the ophthalmic toxicity of DFO, we validated the pathological features observed in the clinic by intraperitoneally injecting C57BL/6J wild-type mice with a dose of 100 mg/kg DFO three times a week from weaning age onwards. Fundus screening by SW-AF was conducted to identify any changes concomitant with the administration of DFO. We found a progression of the spotting presentation within three months of DFO injection in mice (Fig 2A–C). Hyper-autofluorescent spots were less notable in the mice injected with DFO for less than one month (Fig 2A). Fundus spotting became pronounced as the administration of DFO continued
Referência(s)