FTO was increased in Aβ induced RPE degeneration
To identify whether Aβ1-40 treatment alters the expression of m6A regulators in RPE cells, we measured the levels of several major RNAs associated with m6A methylation (METTL3 and METTL14), demethylation (FTO and ALKBH5) and recognition (YTHDF1, YTHDF2 and YTHDF3). The primary mouse RPE cells were cultured with 2.5μM Aβ1-40 for 24 hours. The result of quantitative real-time PCR (qRT-PCR) shown that FTO was up-regulated (2.06±1.38-fold) in Aβ group, compared with phosphate-buffered saline (PBS) group (Figure 1a). We further established the intravitreally injected Aβ1-40 mice and detected the mRNA level of m6A modification RNAs in the RPE–choroid complex at day 4 (Figure 1b). qRT-PCR data revealed that expression of FTO was increased (1.53±0.28-fold) at RNA levels as compared to its respective controls. To investigate whether Aβ1-40 affects FTO protein expression in vivo, FTO expression in RPE–choroid complex was assessed at different time points exposed to Aβ (Figure 1c). In line with initial observation, notable increases of FTO were observed at day 2, day 4, and day 6, with the expression peaked at day 4. Therefore, we chose day 4 for subsequent studies. These findings suggest that Aβ1-40 can induce FTO transcription and expression in RPE cells.
To assess whether the Aβ1-40 deposition induces RPE cell impairment, we used fundus photography (FP) to exam the morphology of fundus in Aβ mice at day 4 (Figure 1d). In contrast to vehicle-injected eyes, the fundus of Aβ mice exhibited areas of depigmentation indicating RPE atrophy and underlying photoreceptor degeneration. This finding suggests that Aβ1-40 deposition can spread through the retina and induce RPE degeneration. Next, to investigate the potential role of FTO in Aβ1-40-mediated RPE degeneration model, we analyzed immunofluorescence staining of Aβ-mice retinal sections and found that FTO showed strong fluorescence signal in the nucleus and perinucleus of RPE cells, whereas slight nuclear signaling was observed in the PBS group (Figure 2a). For further confirmation, primary mouse RPE cells exposed to Aβ1-40 for 24 h were analyzed with immunofluorescence staining. FTO fluorescence signal was increased in the Aβ1-40 group, and mainly concentrated in the nucleus and perinucleus area, compared with PBS group (Figure 2b). Collectively, these data established that Aβ1-40-induced overexpression of FTO could be an important mediator in RPE degeneration.
Inhibition of FTO exacerbated RPE degeneration in Aβ-induced damaging model
To investigate the beneficial function of FTO during Aβ1-40 induced RPE degeneration, we chose the sodium form of Meclofenamic acid (MA-1) as FTO specific inhibitor [27] and measured cell viability of primary mouse RPE cells cultured with different concentration of MA1 at 24 h (Figure 3a). The cell viability of RPE cells in 50μM MA1 group was 101±3.5% and 100μM MA1 group was 97.1±1.8%, showing no significant difference with PBS group. We further detected FTO expression in RPE cells exposed to various concentrations of MA1 at 24 h (Figure 3b). We identified that the treatment of 200μM MA1 resulted in significant decreases in the FTO expression levels. Considering the above factors, we chose 100μM MA1 for subsequent studies and validated the inhibition effect of MA1 in Aβ-induced damaging model by Western blot (Figure 3c). The protein level of FTO significantly decreased in Aβ/MA1 group, compared with PBS group and Aβ group. Furthermore, we performed intravitreal injection (IV) experiment in BALB/c mice with a mixture of 87.5μM Aβ1-40 and 300μM MA1 as IV-Aβ/MA1 mice and detected protein level of FTO in its RPE–choroid complex (Figure 3d). Western blot data shown a significant FTO-attenuating effect in IV-Aβ/MA1 mice in comparison to IV-Aβ mice. Taken together, our results indicated that MA1 effectively inhibited Aβ-induced FTO upregulation in RPE cells.
To clarify the relationship between RPE degeneration and FTO expression, FP and ocular coherence tomography (OCT) were used to assess the fundus and retina of IV-PBS mice, IV-Aβ mice, IV-Aβ/MA1 mice and IV-MA1 mice (Figure 3e). While the fundus of IV-MA1 mice and IV-Aβ mice showed slight and moderate depigmentation comparing to IV-PBS mice, augmented depigmentation was observed in the fundus of IV-Aβ/MA1 mice, indicating that Aβ treatment induced RPE degeneration was aggravated by FTO inhibition. OCT identified no significant changes between IV-PBS mice and IV-MA1 mice. However, IV-Aβ/MA1 significantly aggravated retinal structure disorder, compared to IV-Aβ, confirming that the inhibition of FTO expression exacerbated RPE degeneration in Aβ-induced damaging model. These results suggest that FTO may play a protective role in IV-Aβ mice by rescuing RPE degeneration.
m6A-mRNA Epi-transcriptomic microarray identified PKA as a downstream target of FTO
To investigate the molecular mechanism of FTO and identify its downstream targets in Aβ induced RPE degeneration, we performed m6A-mRNA Epi-transcriptomic Microarray to detect m6A levels of RNA extracted from RPE-choroid complex in Aβ mice and PBS mice. The volcano plot displayed that, m6A levels of 571 genes were differentially methylated among 37739 transcripts detected in the microarray (Figure 4a). 78.3% mRNAs (447 transcripts) were demethylated (green point) and 21.7% mRNA (124 transcripts) were methylated (red point). Since the increase of other m6A writers and readers could not fully explain the decrease in m6A, we suggest that FTO overexpression may result in the m6A demethylation in Aβ1-40 induced RPE degeneration.
Gene Ontology (GO) analysis report revealed that Aβ1-40 induced differential modified m6A RNAs mainly enriched in biological regulation of biological process, binding of molecular function, cell and cell part of cellular component (Figure 4b). Among these, we selected protein kinase A (PKA), a phosphorylase in cAMP/PKA/ cAMP-response element-binding protein (CREB) signaling pathway as a candidate target of FTO-mediated m6A modification for further investigation. Microarray identified that m6A level of PKA was downregulated in Aβ mice, and pathway analysis report suggested that cAMP/PKA/CREB signaling pathway was involved in regulation of above biological process (Figure 4c). PKA was activated by cAMP level and could phosphorylate CREB, a nuclear transcription factor, to its active state, promoting gene transcription and translation, such as brain derived neurotrophic factor (BDNF) (Figure 4d). To verify m6A level of PKA mRNA in Aβ mice, we performed m6A-RNA binding protein immunoprecipitation (MeRIP). qPCR analyses of MeRIP enriched RNA from Aβ mice confirmed that PKA transcripts were hypomethylated in Aβ mice (Figure 4e), suggesting that FTO overexpression may result in PKA mRNA hypomethylation. Taken together, these findings hinted that PKA was a downstream target of FTO.
FTO suppressed PKA transcription and promoted translocation
To investigate the effect of FTO on PKA expression, we measured FTO and PKA protein level in Aβ mice and IV-Aβ/MA1 mice. While the expression of FTO was significantly increased in Aβ mice, PKA was decreased in comparison to the controls (Figure 5a). When the expression of FTO was inhibited by MA1 in IV-Aβ/MA1 mice, protein level of PKA was reversed, comparing to IV-Aβ mice (Figure 5b). Later, we confirmed the regulatory effect of FTO on PKA expression in vitro (Figure 5c). PKA was decreased in RPE cells of Aβ group comparing to PBS group. Meanwhile, the expression level of PKA was higher in Aβ/MA1 group compared to Aβ group. These results show that PKA was significantly suppressed by FTO in Aβ-induced RPE degeneration model and this suppression could be reversed by MA1 treatment. Next, we used confocal microscopic analysis to detect the immunofluorescence of FTO and PKA expression. FTO and PKA showed significant overlaid fluorescence signal around the nuclei of RPE cells in Aβ mice, whereas limited FTO and PKA signaling was observed in the PBS group (Figure 5d). Strong immunofluorescence of PKA was observed in the nuclear of primary RPE cells exposed to Aβ for 24h (Figure 5e), indicating that FTO regulated not only PKA’s expression, but also PKA’s location. Taken together, these results indicate that PKA expression and translocation can be triggered by FTO overexpression.
Aβ induced FTO expression regulated PKA/CREB signaling pathway
To investigate whether FTO mediated PKA/CREB signaling pathway, we detected the mRNA level of FTO and factors of PKA/CREB signaling pathway in Aβ mice (Figure 6a). qRT-PCR showed that FTO expression was increased at mRNA level in Aβ mice, but PKA showed slightly decrease without statistical significance. Downstream targets CREB and BDNF were significantly downregulated. We further evaluated the protein level of FTO and PKA/CREB signaling pathway by Western blot in Aβ mice (Figure 6b). Consistently, a significant increase of FTO was observed, while others were decreased, suggesting that FTO induced PKA down-regulation would result in the suppression of PKA/CREB signaling pathway. To confirm the regulatory role of FTO in PKA/CREB signaling pathway, we established FTO-inhibition model in vivo, and detected the mRNA level of FTO and factors of PKA/CREB signaling pathway (Figure 6c). qPCR data showed that inhibiting FTO reduced the mRNA of FTO and PKA, but promoted mRNA expression of downstream CREB and BDNF, comparing to IV-Aβ mice. We further validated the regulatory effect of FTO through Western blot (Figure 6d). Compared to IV-Aβ mice, suppression of FTO significantly increased the protein levels of PKA, p-CREB and the precursor of BDNF (pro-BDNF), demonstrating that FTO inhibition significantly abolished the suppression of PKA/CREB signaling pathway.
We further validate the effect of FTO on PKA/CREB signaling pathway in vitro. qRT-PCR results of RPE cells from Aβ group showed that FTO was increased at mRNA level as well as PKA and CREB but BDNF showed slightly decrease without statistical significance, comparing to PBS group (Figure 6e). qRT-PCR results of RPE cells from Aβ/MA1 group showed significantly reduction in mRNA levels of FTO, PKA and BDNF, but an increase in CREB, comparing to Aβ group (Figure 6f). Next, we performed Western blot and found that downregulation of FTO caused upregulation of PKA, p-CREB and pro-BDNF (Figure 6g). Taken together, these results indicated that the FTO dependent protective effects on RPE survival and repair was mediated via PKA/CREB signaling pathway.