IGF2BP2 Maintains Retinal Pigment Epithelium Homeostasis by Stabilizing PAX6 and OTX2

Abstract

Purpose: N ⁶-methyladenosine (m⁶A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N⁶-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m⁶A modifications in maintaining the characteristics of RPE cells. m⁶A readers play a crucial role in executing the functions of m⁶A modifications, which prompted our investigation into their regulatory roles in the RPE.

Methods: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, β-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA–mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography.

Results: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo.

Conclusions: Our data suggest a crucial role of IGF2BP2 as an m⁶A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.

The retinal pigment epithelium (RPE) is a monolayer epithelial cell sheet located in the outermost layer of the retina.^1,2 It serves various important functions, such as light absorption, epithelial transport, secretion of growth factors, participation in the visual cycle, phagocytosis of discarded outer segments of photoreceptors, and establishment of the blood–retinal barrier.^3–5 RPE dysfunction leads to the pathogenesis and progression of retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and even vision loss.^6–9 

Extensive research has focused on transcription factors that play indispensable roles in the development and maintenance of RPE cell characteristics.^10–14 D'Alessio et al.¹⁵ previously reported that transduction of foreskin fibroblasts with PAX6, OTX2, MITF, SIX3, GLIS3, and FOXD1 could induce cell transformation into RPE, underscoring the significance of these genes in RPE cell differentiation and identity maintenance. PAX6 collaborates with MITF/TEFC to ensure proper development of the RPE,¹⁰ and downregulation of PAX6 expression decreases melanin pigmentation in the RPE.¹⁶ OTX2 knockdown leads to a reduction in RPE-specific functions, such as melanogenesis and retinol metabolism, and activates stress signaling pathways and inflammatory genes.¹⁷ MITF, a crucial marker gene of RPE cells, participates in melanin biogenesis and antioxidant stress responses.^18,19 The absence of MITF leads to transformation of the RPE into the retina, resulting in degenerative changes in the retina.^19,20 

N ⁶-methyladenosine (m⁶A) mRNA modifications play a critical role in various biological processes²¹. Reversible m⁶A methylation is mediated by m⁶A modifiers, primarily methyltransferases (METTL3, METTL14, and WTAP), demethylases (FTO and ALKBH5), and readers (YTHDC1/2, YTHDF1/2/3, and IGF2BP1/2/3), which are responsible for recognizing methylated sites.^22,23 m⁶A readers are proteins that specifically recognize and bind to the m⁶A modification on RNA. Depending on the specific readers recognizing m⁶A modifications, m⁶A modification serves different biological functions, mainly altering mRNA transcription splicing, enhancing the translation of specific RNAs, or modifying the stability of these RNAs.²³ Mounting evidence suggests the involvement of m⁶A modifications and their modulators in AMD pathogenesis.^24–26 However, there is a lack of studies on the regulation of RPE cell characteristics by m⁶A modification. 

In the current study, we found that insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) regulates RPE homeostasis by recognizing m⁶A modifications. IGF2BP2 directly modulates PAX6 and OTX2 mRNA stability as an m⁶A reader, and IGF2BP2 deficiency attenuates the antioxidant stress response in the RPE, contributing to cellular aging and inflammation. Consistent with in vitro findings, adeno-associated virus (AAV)-shIGF2BP2 in the RPE adversely affects visual function, culminating in degenerative changes in the retina in vivo. 

Materials and Methods

Clinical Materials

The human eye tissues were obtained from deceased donors who passed away unexpectedly and did not have any known ocular diseases (the health of the eyes was confirmed through examination as the anterior segment tissues were used for transplantation), adhering to the Ethics Committee of the Zhongshan Ophthalmic Center of Sun Yat-Sen University (2023KYPJ231). The research was conducted adhering to the tenets of the Declaration of Helsinki. The donors’ information is available in Supplementary Table S1. 

Cell Culture

The eye cup was initially immersed in dispase solution and incubated at 37°C for 40 minutes. Subsequently, the retina was dissected from the optic nerve, and the RPE layer was gently separated from the eye cup. The RPE layer was then transferred to 0.05% trypsin–EDTA solution and incubated at 37°C for 10 minutes. Ultimately, RPE cells were seeded onto cell culture dishes precoated with 10% Matrigel and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.²⁷ Reagent information is provided in Supplementary Table S1. 

Histopathology and Immunofluorescence Staining

RPE and retinal flatmounts were fixed in 4% polyformaldehyde for 15 minutes at room temperature, and mouse eyeballs were fixed with 10% formalin for 3 hours. Subsequently, mouse eyeballs were fixed, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, after paraffin embedding and deparaffinization, tissue sections were subjected to antigen retrieval, followed by permeabilization and blocking using a solution containing 0.3% Triton X-100 and 3% bovine serum albumin. Subsequently, the samples underwent overnight incubation with primary antibodies under conditions maintained at 4°C. The next day, secondary antibodies were incubated for 1 hour. The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Antibody information is provided in Supplementary Table S2. 

RNA Interference

Two short-hairpin RNAs (shRNAs) targeting IGF2BP2, IGF2BP3, YTH N⁶-methyladenosine RNA binding protein 1 (YTHDF1), YTHDF2, YTHDF3, and YTHDC1 were cloned into the PLKO.1 lentiviral plasmid. RPE cells underwent transfection with lentiviral particles encoding shRNAs for approximately 24 hours, followed by selection with 1.5- µg/mL puromycin for 48 hours. After drug screening, the cells were passaged once, and the transduction efficiency was ultimately validated by quantitative PCR (qPCR). Scrambled shRNA, which did not target any known gene, was employed as a negative control. The shRNAs employed in our study are provided in Supplementary Table S3. 

Phagocytosis Assay

Photoreceptor outer segments (POS) were isolated from porcine eyes and labeled by fluorescein isothiocyanate (FITC) according to a protocol outlined by Parinot et al.²⁸ Preceding the commencement of the cellular phagocytosis assay, RPE cells were passaged onto 0.4- µm Transwell inserts (3470; Corning, Corning, NY, USA) coated with 10% Matrigel. Before POS were added, primary RPE cells were pretreated with 30% FBS for 1 hour to induce phagocytosis in RPE cells.²⁹ Then, 10⁶ FITC-labeled POS were added to RPE cells and incubated for 1 hour at 37°C. The unbound POS were removed by washing with PBS before fixation. Image J (National Institutes of Health, Bethesda, MD, USA) was used for POS quantification. We applied a double-blinding method to ensure that the experimental results were unbiased. 

MitoTracker

Cells were treated with 100-nm/mL MitoTracker Red CMXRos dye (Thermo Fisher Scientific, Waltham, MA, USA), and the incubation was carried out at 37°C for 30 minutes. Post-incubation, the cells were fixed with paraformaldehyde at –20°C for 20 minutes. The specimens were examined under a LSM 800 microscope (ZEISS, Oberkochen, Germany). 

Cellular Reactive Oxygen Species Assay

RPE cells were dissociated into single cells using 0.05% trypsin EDTA. After centrifugation and resuspension, 2′,7′-dichlorofluorescin diacetate (DCFDA) dye was added to the cells, followed by an incubation at 37°C for 30 minutes. Subsequently, the cells were analyzed using flow cytometry. 

qPCR and RNA Decay Assays

For RNA decay assays, RPE cells were exposed to actinomycin D or dimethyl sulfoxide (DMSO), and total RNA was collected at the 2 and 4 hours post-treatment. Total RNA was isolated from cells using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). We employed the PrimeScript RT Master Mix Kit (Takara Bio, Shiga, Japan) to reverse transcribe RNA into cDNA. qPCR was conducted using the iTaq Universal SYBR Green Supermix Kit (Bio-Rad, Hercules, CA, USA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization control. A list of the primer used is provided in Supplementary Table S4. 

HITS-CLIP Experiment

The high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) experiment was based on a protocol described by Moore et al.,³⁰ with slight modifications. Briefly, RPE cells were transfected with lentivirus particles encoding FLAG-IGF2BP2. Cells were ultraviolet crosslinked by irradiation once at 400 mJ cm² and then again at 200 mJ cm². Cells were then collected and lysed, and cellular RNA was fragmented. Subsequently, the fragmented RNA was incubated with an anti-FLAG antibody, and then antibody–RNA complexes were recovered through immunoprecipitation. After purification by transfer to a nitrocellulose membrane, the antibody–RNA complexes were eluted using proteinase K. The RNA was further purified and subjected to sequencing. 

HITS-CLIP Data Analysis

The HITS-CLIP assay was based on a protocol described by Moore et al.³⁰ The raw reads underwent quality assessment using FASTQC 0.0.2. The HITS-CLIP reads aligned to the hg19 human reference genome by using the Burrows–Wheeler Aligner (BWA) tool. For mutation site identification, crosslink-induced mutation sites (CIMS) software and the Perl tool were utilized to search mutation sites in the BWA alignment and tag sequences, selecting robust CIMS with a false discovery rate (FDR) of ≤ 0.001. The nucleotides spanning −10 to +10 of the CIMS were obtained in the Browser Extensible Data (BED) file format for subsequent motif analysis. MEME 5.5.5 was used for the de novo motif analysis with P ≤ 0.01.³¹ 

RNA-Seq Data Analysis

The raw reads underwent quality assessment using FASTQC 0.11.5³² and were subsequently trimmed to eliminate adaptor sequences using Trimmomatic 0.39 tools. RNA-seq reads were aligned to the hg19 human reference genome using STAR software,³³ and the calculation of transcripts per million (TPM) values was carried out using the RNA-seq by expectation-maximization (RSEM) tool.³⁴ Differentially expressed genes were identified using the DESeq2 R package (R Foundation for Statistical Computing, Vienna, Austria). The criteria for selection included a log₂ fold change of ≥1 and a FDR of <0.05.³⁵ We employed the online software Metascape (www.metascape.org) for Gene Ontology (GO) enrichment analysis. The parameters were set with cutoffs of P = 0.01 and q = 0.05.³⁶ 

Animal Model and Treatment

The design and experimental protocols for animal experiments were approved by the Animal Experiment Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-Sen University (W2023006). C57BL/6 mice (6–8 weeks old, 18–23 g in weight) were purchased from the Guangdong Laboratory Animal Center (Guangzhou, China) and maintained in the animal facility of the Zhongshan Ophthalmic Center at Sun Yat-Sen University (Guangzhou, China). Mice were housed under a 12-hour light/dark cycle and fed ad libitum. After anesthesia, 1 µL CNV-ZSGREEN-AAV2-U6-shIGF2BP2 (10¹¹ viral particles/µL) was injected into the subretinal space, and the paired eye was injected with negative control AAV. 

Optical Coherence Tomography Evaluation

An intraperitoneal injection of 0.6% pentobarbital sodium was administered for mouse anesthesia (100 µL/10 g). Pupils were dilated using tropicamide ophthalmic drops, and corneas were lubricated with hypromellose gel. Optical coherence tomography (OCT; Heidelberg Engineering, Heidelberg, Germany) was performed on both eyes to investigate structural changes. Heidelberg Eye Explorer software was used to compute retinal thickness and volume. 

Electroretinography Testing

Prior to the electroretinography (ERG) test, recorded using a Celeris rodent ERG device (Diagnosys, Lowell, MA, USA), mice underwent overnight dark adaptation. After the mice were anesthetized, pupil dilation was performed, and the surface of the eyes was kept moist. Electrodes were positioned atop each cornea, and scotopic and photopic ERG responses were subsequently recorded. The light-adapted ERG was conducted subsequent to dark-adapted ERG. Following a 10-minute light adaptation (30 cd/m²; white, 6500K), photopic ERG was executed with flash intensities ranging from 0.3 to 30 cd s/m². The C-waves followed a 100-ms light stimulus (150 cd/m²; white, 6500K). 

Western Blot Analysis

The RPE–choroid tissues, retina, and RPE cells were lysed in 100 µL radioimmunoprecipitation assay (RIPA) buffer and supplemented with protease inhibitor cocktails and protein phosphatase inhibitors. Tissues were also sonicated. For the western blotting analysis, 20 to 50 µg of total protein was used. 

Statistical Analysis

Prism 6 (GraphPad, Boston, MA, USA) was used for the statistical analyses. The unpaired two-tailed Student's t-test was used to determine the significance of differences between groups. For multiple condition comparisons, Šídák’s multiple comparisons test was used. Data are presented as means ± SD. Details on replicates performed for each experiment can be found in the figure legends. 

Results

IGF2BP2 Modulates RPE Phagocytosis

Phagocytosis is a major physiological function of the RPE³⁷; therefore, phagocytic ability was used as an observational parameter to investigate the effects of m⁶A regulation on RPE cells. Primary cultured RPE cells were characterized by positive staining for RPE65, PAX6, MITF, OTX2, and ZO-1 (Supplementary Fig. S1A). m⁶A-related reader genes, including IGF2BP2, IGF2BP3, YTHDC1, YTHDF1, YTHDF2, and YTHDF3, were knocked down by shRNA transfection (Supplementary Fig. S1B). The phagocytic function was assessed using the experimental scheme outlined in Figure 1A.²⁹ Z-stack fluorescence imaging revealed the intracellular localization of fluorescence-labeled POS, confirming the phagocytic capability of ex vivo cultured RPE cells (Fig. 1B). We found that IGF2BP2 was the most effective m⁶A reader affecting RPE phagocytosis among the candidates, with the number of phagocytosed POS being only 56.6% of those in the control group (Figs. 1B, 1C), suggesting that IGF2BP2 is a principal m⁶A-related player in the regulation of RPE cell function. 

Figure 1.

 

IGF2BP2 controlled RPE phagocytosis. (A) Experimental scheme for detecting the phagocytic function of RPE cells. (B) FITC-labeled POS were visualized in RPE cells transduced with scrambled shRNA-(SCR), shIGF2BP2, shIGF2BP3, shYTHDC1, shYTHDF1, shYTHDF2, and shYTHDF3 (n = 3 biological replicates). Scale bar: 50 µm. (C) Quantification of POS in RPE cells for the indicated transfection. For each group, five regions were randomly selected. *P < 0.05 (two-tailed unpaired t-test; n = 3 for each group).

IGF2BP2 Affects Cellular Function by Regulating the mRNA Stability of PAX6 and OTX2

To further gain insight into the influence of IGF2BP2 on the RPE, we compared the transcriptional profiles of IGF2BP2 knocked-down RPE cells (shIGF2BP2-RPE group) with those of control RPE cells (Supplementary Fig. S1B). Principal component analysis indicated that the excellent intragroup sample reproducibility and gene expression pattern in the shIGF2BP2-RPE group were quite distinct from those in the control group (Supplementary Fig. S2A). A total of 786 and 472 mRNAs were downregulated and upregulated, respectively, in the shIGF2BP2-RPE group. Notably, the loss of IGF2BP2 resulted in the downregulation of crucial genes that maintain the characteristics of the RPE, such as MITF, PAX6, and OTX2. Moreover, the expression of a series of genes that are linked to the biological processes of the retinoid cycle (RARRES2, RBP1, CRABP2), secretion (SERPINF1), phagocytosis (MYO7A, GULP1, RAP1GAP), and pigmentation (MYO7A, SPNS2) (Fig. 2A) were decreased in the shIGF2BP2-RPE group. Consistent results were obtained using GO and Gene Set Enrichment Analysis (GSEA) analyses (Fig. 2B, Supplementary Fig. S2B).³⁸ 

Figure 2.

 

Gene regulatory network analysis identified IGF2BP2 controlling RPE cell characteristics. (A) Heatmap demonstrated normalized transcripts per million reads (TPM) values of the top 100 downregulated genes in the shIGF2BP2-RPE group, with the addition of the gene list of Supplementary Table S5. (B) GO analysis of downregulated genes in the shIGF2BP2-RPE group. (C) Pie charts depicting the proportions of peak distributions in the 5′ UTR, CDS, and 3′ UTR regions across the CIMS. (D) Metagene profiles of CIMS distribution across the 5′ UTR, CDS, and 3′ UTR regions in RPE cells. (E) Each sequence motif was identified within m⁶A peaks by MEME analysis of the CIMS. (F) Venn diagram indicates the overlapping and unique genes for the CIMS containing the m⁶A peak and RNA-seq downregulated genes. (G) GO analysis of the common elements between the CIMS containing the m⁶A peak and RNA-seq downregulated genes. (H) Genome browser tracks for the indicated CIMS and m⁶A peak signals across the PAX6 and OTX2 loci. (I) Representative mRNA profile of PAX6 and OTX2 at indicated time points after actinomycin D (ActD) treatment (5 µg/mL) in RPE cells treated by DMSO and STM2457 for 24 hours (10 µm). The qPCR values were normalized to the values in the cells for the 0-hour time point. *P < 0.05 (two-tailed unpaired t-test; n = 3 for each group). (J) Representative mRNA profile of PAX6 and OTX2 at indicated time points after ActD treatment (5 µg/mL) in the scrambled shRNA-(SCR) and shIGF2BP2-RPE groups. The qPCR values were normalized to the values in the cells for the 0-hour time point. *P < 0.05, **P < 0.01 (two-tailed unpaired t-test; n = 5 for each group). (K) Western blot analysis of IGF2BP2, PAX6, OTX2, and MITF protein levels with the indicated treatments. (L) Quantification of western blot analysis; values were normalized to the values in the control cells. *P < 0.05, ***P < 0.001 (two-tailed unpaired t-test; n = 3 for each group).

IGF2BP2, an m⁶A reader, was previously reported to enhance RNA stability by recognizing m⁶A modifications in RNA.^22,23 To gain a deeper understanding of how IGF2BP2 affects the expression of characteristic genes in RPE cells, HITS-CLIP assays were performed.³⁰ The HITS-CLIP results identified IGF2BP2-binding RNAs and their specific binding sites. Among the CIMS, 41.7% were found to be distributed across the 5′ untranslated regions (UTR), coding sequences (CDSs), and 3′ UTR (Fig. 2C). Most sites were concentrated around the start and stop codons, as well as the 3′ UTR (Fig. 2D). m⁶A is a co-transcriptional modification that occurs in the sequence context RRACH (R = A or G; H = A, C, or U; A = m⁶A).³⁹ Motif enrichment analysis of the m⁶A-containing peaks was performed using CIMS, which revealed three distinct consensus motifs centered on the AC core of the m⁶A motif, which was significantly enriched (Fig. 2E). A total of 5335 RNAs associated with these three motifs intersected with genes that were downregulated in the shIGF2BP2-RPE group, and 180 overlapping genes were identified (Fig. 2F). GO analysis of these genes revealed a decline in cell fate specification, pigmentation, secretion by cells, and response to retinoic acid (Fig. 2G). Analysis of the HITS-CLIP assay results revealed m⁶A modifications at the sites where IGF2BP2 binds to OTX2 and PAX6 (Fig. 2H). Treatment of RPE cells with the METTL3 inhibitor STM2457⁴⁰ led to decreased stability of OTX2 and PAX6 (Fig. 2I), suggesting regulation of PAX6 and OTX2 mRNA stability by m6A modification. Moreover, inhibition of IGF2BP2 binding to m⁶A modification sites using CWI1-2⁴¹ resulted in a notable reduction in PAX6 and OTX2 expression levels (Supplementary Fig. S2C). RNA decay assays further demonstrated that IGF2BP2 knockdown significantly diminished the stability of PAX6 and OTX2 mRNA in RPE cells (Fig. 2J), highlighting the recognition of m6A modifications on mRNA by IGF2BP2, thereby modulating their stability. Moreover, we observed that the downregulation of IGF2BP2 and PAX6 expression was accompanied by a decrease in MITF expression (Figs. 2K, 2L). Therefore, we propose that IGF2BP2 influences these regulatory networks by affecting the functionality and homeostasis of RPE cells, thereby ensuring proper physiological function through the recognition and stabilization of essential transcription factors. 

Knockdown of IGF2BP2 Induces Senescence of RPE Cells

Notably, 5-ethynyl-2′-deoxyuridine (EdU) staining of cells indicated a decrease in cell proliferation after IGF2BP2 knockdown (Supplementary Figs. S3A, S3B). Meanwhile, RNA-seq data revealed that the upregulated genes following IGF2BP2 knockdown are enriched in cellular aging, suggesting a permanent cell-cycle arrest in RPE cells (Fig. 3A, Supplementary Figs. S3C, S3D). Beta-galactosidase staining indicated a significant increase in the proportion of senescent cells in the IGF2BP2 knockdown group (Figs. 3B, 3C). Strikingly, cellular reactive oxygen species (ROS) levels were elevated in the shIGF2P2-RPE cells (Fig. 3D), and downregulation of IGF2BP2 expression led to mitochondrial fission, which indicated low mitochondrial membrane potential (MMP) (Fig. 3E).⁴² Reduced MMP suggests potential damage to the mitochondria, which implies the release of more cellular ROS. Thus, these results indicate that IGF2BP2 knockdown led to cellular oxidative stress and triggered senescence. 

Figure 3.

 

Knockdown of IGF2BP2 induced cellular senescence. (A) GO analysis of upregulation genes in the shIGF2BP2-RPE group. (B) Image of RPE cells stained with IGF2BP2 (n = 3). Scale bar: 50 µm. (C) Quantification of beta-galactosidase–positive RPE cells for the indicated transfections. For each group, five regions were randomly selected. **P < 0.01 (two-tailed unpaired t-test; n = 3 for each group). (D) ROS production levels in the control group and shIGF2BP2-RPE group measured by flow cytometry (n = 3). (E) MitoTracker fluorescence was detected in the control and shIGF2BP2-RPE groups (n = 3). Scale bar: 10 µm.

IGF2BP2 Deficiency Impairs RPE Function

To explore the in vivo regulatory role of IGF2BP2 in the RPE, we initially verified the expression of IGF2BP2 in RPE–choroid tissues using immunofluorescence staining (Fig. 4A). We then used an AAV containing the shIGF2BP2 sequence with a ZSGREEN reporter to suppress IGF2BP2 expression in mouse RPE by subretinal injection (Fig. 4B, Supplementary Fig. S4A). Using whole-mount ZSGREEN imaging of RPE cells, we observed the successful transfection of RPE cells (Supplementary Fig. S4B). In comparison with the control group, the AAV-shIGF2BP2–infected group exhibited a substantial reduction in IGF2BP2 mRNA and protein levels in the RPE (Fig. 4C, Supplementary Fig. S4C), whereas IGF2BP2 expression in the retinal layers did not change (Supplementary Fig. S4D). 

Figure 4.

 

Knockdown of IGF2BP2 affected mice RPE identity. (A) Fluorescence image of IGF2BP2 and F-actin in RPE–choroid tissues. Scale bar: 10 µm. (B) Animal experimental scheme. Negative control (NC) AAV or AAV-shIGF2BP2 was delivered into the subretinal space of 6- to 8-week-old mice. (C) qPCR shows mRNA expression of RPE cells at 10 days after injection. ***P < 0.001 (two-tailed unpaired t-test; n = 6 mice). (D) Quantification of cell number from F-actin fluorescence image at 10 days. ***P < 0.001 (two-tailed unpaired t-test; n = 5 mice). Scale bar: 10 µm. (E) Rhodopsin fluorescence was detected as an indicator of phagocytic function of RPE cells in the morning (9:00 AM) and afternoon (4:30 PM) at 10 days after injection (n = 3 mice). Scale bar: 10 µm. (F) Immunostaining image of PAX6, OTX2, MITF, and RPE65 in the posterior eye segments after injection for 10 days (n = 3 mice). Scale bar: 50 µm. (G) qPCR presenting mRNA expression of RPE cells at 10 days after injection. qPCR values were normalized to the values in control cells. The genes validated by qPCR were selected based on their differential expression from RNA-seq data following the knockdown of IGF2BP2 in cultured RPE cells.*P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test; n = 4 or 5 mice).

Subsequently, the F-actin dye phalloidin was used to assess RPE morphology in flatmounted retinal tissues at 10 days post-virus injection. Compared to the RPE layer of the control group, which exhibited a regular hexagonal configuration, there was a notable increase in cell size and aberrant cell morphology in the shIGF2BP2-RPE group (Fig. 4D). Functional tests of RPE phagocytosis revealed that ex vivo RPE in the knockdown group showed decreased adherence to the outer segments of photoreceptors compared with that in the control group in the morning (9:00 AM). There was an increase in intracellular and extracellular outer segments (labeled with opsin) over time in the shIGF2BP2 group, whereas the control group showed a decrease in outer segments over time (Fig. 4E). These results suggest a diminished ability of the RPE in the knockdown group to phagocytose and digest photoreceptor outer segments. 

Consistent with previous findings in vitro, fluorescence staining and quantitative qPCR showed that knockdown of IGF2BP2 led to downregulation of RPE65, PAX6, MITF, and OTX2 expression in the shIGF2BP2-RPE group of mice (Figs. 4F, 4G). Moreover, mRNA expression of RBP1, LRAT, RDH5, TYR, MYO7A, GULP1, and APPL2, which encode essential RPE-specific proteins involved in the visual cycle, pigmentation, and phagocytosis, was also reduced in the AAV-shIGF2BP2-RPE group (Fig. 4G). These data suggest a pivotal role for IGF2BP2 in maintaining the function and characteristics of RPE cells. 

Knockdown of IGF2BP2 Affects Visual Function In Vivo

The RPE is critical for maintaining retinal homeostasis, and retinal photoreceptor cells tend to degenerate or even die when RPE function declines, ultimately resulting in retinal atrophy.^43,44 To elucidate visual dysfunction in response to IGF2BP2 suppression, shIGF2BP2 mice were subjected to ERG measurements 1, 2, and 4 weeks after AAV injection. The C-wave was significantly reduced at each time point, indicating impaired RPE function.⁴⁵ Notably, the C-wave in mice RPE with IGF2BP2 knockdown exhibited a significant decrease over time (Figs. 5A, 5B). Scotopic ERG responses also significantly declined over time in shIGF2BP2 mice, indicating impaired photoreceptor phototransduction (A-wave) and rod bipolar cell depolarization (B-wave) (Figs. 5C–5E). 

Figure 5.

 

Knockdown of IGF2BP2 diminished electrical activity of the mice retina. (A) ERG-recorded C-waves at 2 weeks after AAV vector injection. (B) Quantification of C-wave ERG amplitude following a 100-ms light stimulus (150 cd/m²; white, 6500K). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired two-tailed Student's t-test; n = 10 mice 1 and 2 weeks old and n = 6 mice 4 weeks old). (C) ERG-recorded scotopic A-waves and B-waves at 1, 2, and 4 weeks after injection. (D) Quantification of A-wave ERG amplitudes. ns, not significant; *, †, ‡, and § symbols indicate statistical significance determined using the Holm–Šídák method, with alpha = 5.000% (multiple t-tests; n = 6 mice 1 week old, n = 8 mice 2 weeks old, and n = 8 mice 4 weeks old). Black line indicates 1-week-old NC versus 1-week-old shIGF2BP2; orange line indicates 2-week-old NC versus 2-week-old shIGF2BP2; green line indicates 4-week-old NC versus 4-week-old shIGF2BP2; blue line indicates 1-week-old shIGF2BP2 versus 2-week-old shIGF2BP2. (E) Quantification of B-wave ERG amplitudes. ns, not significant; *, †, ‡, and § symbols indicate statistical significance determined using the Holm–Šídák method, with alpha = 5.000% (multiple t-test; n = 6 mice 1 week old, n = 8 mice 2 and 4 weeks old). Black line indicates 1-week-old NC versus 1-week-old shIGF2BP2; orange line indicates 2-week-old NC versus 2-week-old shIGF2BP2; green line indicates 4-week-old NC versus 4-week-old shIGF2BP2; blue line indicates 1-week-old shIGF2BP2 versus 2-week-old shIGF2BP2.

OCT was used to observe changes in retinal structure. The retina in mice injected with AAV-shIGF2BP2 showed progressive thinning over time (Figs. 6A–6C). This finding was corroborated by quantitative topographic volume assessment (Fig. 6D, Supplementary Fig. S5A). H&E staining of the posterior eye segment showed obvious RPE disruption, aberrant photoreceptor morphology, and retinal thinning at 4 weeks after AAV-shIGF2BP2 injection (Fig. 6B). Additionally, because vascular invasion from the choriocapillaris is a crucial indicator of retinal damage, we performed CD18/ITGB2 (IB4) staining in our mouse model. The results revealed the presence of vascular infiltration within the RPE–choroid complex in the IGF2BP2 knockdown group of mice, which was absent in the control group of mice (Fig. 6E). With the disruption of RPE cell organization and vascular invasion, immunofluorescent staining of Rhodopsin revealed a reduction in photoreceptor cells (indicated by white arrows) (Fig. 6F), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining demonstrated apoptosis of photoreceptor cells in the IGF2BP2 knockdown group (Fig. 6G). Taken together, these results reveal that IGF2BP2 plays a crucial role in maintaining retinal visual function and that the knockdown of IGF2BP2 induces aberrant photoreceptor morphology, retinal thickness thinning, RPE disruption, and photoreceptor cell death. This sequence of events culminates in disturbed retinal homeostasis. 

Figure 6.

 

Knockdown of IGF2BP2 disrupts mice retina homeostasis. (A) OCT scan of mice at 4 weeks. Scale bar: 200 µm. (B) H&E staining of the posterior eye segments after injection for 30 days (n = 5 per group). Scale bar: 100 µm. (C) Quantification of retinal thickness of OCT at 3 and 4 weeks. ns, not significant; asterisk (*) indicates statistical significance determined using the Holm–Šídák method, with alpha = 5.000% (multiple t-test; n = 10 mice). (D) Quantification of retinal volume of OCT at 1 to 4 weeks. ns, not significant; asterisk (*) indicates statistical significance determined using the Holm–Šídák method, with alpha = 5.000% (multiple t-test; n = 10 mice). (E) Fluorescence image of IB4 in RPE–choroid tissues (n = 5 mice). Scale bar: 10 µm. (F) Rhodopsin was detected in the posterior eye segments after injection for 30 days (n = 5 mice). Scale bar: 50 µm. (G) Quantification of TUNEL-positive cell number in posterior eye sections. Scale bar: 50 µm. ***P < 0.001 (two-tailed unpaired t-test; n = 5 mice, for each of which one or two regions were randomly selected).

Discussion

Accumulating evidence supports a significant role for m⁶A modifications in the maintenance of cellular and tissue functions.^23,46,47 Indeed, some readers can enhance RNA stabilization through various mechanisms. Once bound, these proteins may enhance translation, thereby protecting mRNA from degradation; recruit stabilizing proteins that directly prevent decay; or influence mRNA export and localization, ensuring its availability for translation.^21,23 These mechanisms collectively support the longevity and functionality of mRNA within cells. For example, IGF2BP2 recognizes and binds RNA molecules bearing m⁶A modifications through its K homology (KH) domains.^41,48 Our work emphasizes that IGF2BP2 may function as a reader recognizing m⁶A modification sites on PAX6 and OTX2 mRNA. Decreased levels of IGF2BP2 in RPE cells directly or indirectly lead to decreased expression of PAX6, OTX2, and MITF, thereby impacting various functions of RPE cells such as visual circulation and phagocytosis. This disruption ultimately compromises the ability of the retina to maintain its homeostasis and suggests its potential as a target for preventing the occurrence of retinal disease related to RPE malfunction. 

In the in vitro culture of RPE cells, cell density can influence cell polarity, thereby affecting cell function.^4,49 In this study, we found that knockdown IGF2BP2 led to a decrease in the expression of important transcription factors in RPE cells, such as PAX6, OTX2, and MITF, resulting in slower cell proliferation and cell aging. Consequently, the decrease in cell density affected cell polarity and differentiation, partially impacting cell function. This interplay likely represents one of the mechanisms through which IGF2BP2 knockdown affects RPE cell function. Future studies will further investigate the specific effects of IGF2BP2 knockdown on RPE cell polarity and function. RPE dysfunction plays a crucial role in the pathogenesis and progression of retinal diseases.^7,43,50 A reduction in the RPE phagocytic, secretory, and visual cycle is associated with the occurrence of retinal degenerative diseases, such as AMD, retinitis pigmentosa, and Stargardt disease.^4,51–53 Impairment of the RPE barrier function can lead to pathologies such as AMD and macular edema.^4,54 Our in vivo mouse study involving IGF2BP2 knockdown in the RPE showed retinal degeneration, pigment deposition, and choroidal vascular invasion, which align with the characteristic features of AMD. Additionally, knockdown of IGF2BP2 in RPE cells resulted in upregulation of the AMD-associated gene THBS1 and inflammatory factors IL6, IGFBP3, and ICAM1.^55,56 A limitation in our study is that the RNA-seq and CLIP-seq samples were derived from donors of different ages. Consequently, this age discrepancy might introduce variability in interpreting the data. Conducting RNA-seq and CLIP-seq on cross-age paired samples could mitigate this limitation. This finding underscores the critical role of IGF2BP2 in maintaining RPE homeostasis and suggests the need for further exploration of its role in retinal diseases. 

Research into the m⁶A readers, including the YTHDF family and YTHDC2, highlights their diverse and significant impacts on eye-related pathologies and RNA dynamics.²⁶ YTHDF2 has been implicated in RNA degradation and is associated with the regulation of corneal neovascularization.^26,57 YTHDF1/3 has been suggested to play a role in enhanced translation, and YTHDF1, specifically, may play a role in ocular melanoma and oxygen-induced retinopathy.^58,59 YTHDC1 is primarily involved in regulating pre-mRNA splicing and facilitating mRNA nuclear transport, whereas YTHDC2 contributes to mRNA stability and translation.^21,23 Elevated expression of YTHDF2 and YTHDC2 in the serum of patients with Graves’ ophthalmopathy suggests the potential involvement of m⁶A modifications in secondary eye diseases.⁶⁰ HNRNPC/G and HNRNPA2B1 indirectly bind to transcripts that have undergone alternative splicing or processing that results in structural transformations, whereas IGF2BP family readers (IGF2BP1/2/3) primarily enhance mRNA stability and promote mRNA translation.^22,23,61 Currently, there is a paucity of information regarding the regulatory roles of HNRNPC/G, HNRNPA2B1, and IGF2BP1/2/3 in normal and diseased eye tissues. Our study sheds light on the pivotal role of IGF2BP2 in maintaining the normal physiological state of RPE cells and shows that IGF2BP2 knockdown can lead to retinal dysfunction in vivo. 

Acknowledgments

Supported by grants from the National Natural Science Foundation of China (82371082), Guangzhou Science and Technology Program (2024A03J0254/SL2024A03J00493), Young Teacher Training Program of Sun Yat-Sen University (09667-12230012), and Guangdong Basic and Applied Basic Research Foundation (2021A1515010468). 

Disclosure: S. Wu, None; F. Li, None; K. Mo, None; H. Huang, None; Y. Yu, None; Y. Huang, None; J. Liu, None; M. Li, None; J. Tan, None; Z. Lin, None; Z. Han, None; L. Wang, None; H. Ouyang, None