Nrf2 overexpression rescues the RPE in mouse models of retinitis pigmentosa

Abstract

Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2–treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).

AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa

“…These results indicate that the therapeutic effect of Txnip does not require an inhibitory interaction with thioredoxins. This finding is in keeping with previous work which showed that anti-oxidation strategies promoted cone survival in RP mice (Komeima et al, 2006;Wu et al, 2021;Xiong et al, 2015). An additional mutation, S308A, which loses an AMPK/Akt-phosphorylation site on Txnip (Waldhart et al, 2017;Wu et al, 2013), was tested in the context of WT Txnip and in the context of the C247S allele.…”

“…These combinations utilize cell type-specific promoters, reducing the chances of side effects from global expression of these genes. Of note, the Nrf2 expression was limited to the RPE, which we recently showed leads to much improved RPE survival and morphology in RP mice (Wu et al, 2021). Best1-Nrf2 was additive to RedO-Txnip for cone survival.…”

“…These are circumscribed areas without cones that are obvious when the retina is viewed as a flat-mount. AAV-Best1-Nrf2 alone suppressed the formation of these craters (Figure 8A) (Wu et al, 2021), while AAV-RedO-Txnip did not, despite the fact that AAV-RedO-Txnip.C247S provided the most robust RP cone rescue (Figure 2A, Figure 8A,D). It was also noted that dnHIF1α decreased, and Best1-Txnip.C247S.LL351&352AA increased, the FVB-specific retinal craters, while both vectors prolonged RP cone survival (Figure 7A).…”

“…We used a custom MATLAB script to automatically count the number of H2BGFP-positive cones in the central ½ radius of the retina, since RP cones degenerate faster in the central than the peripheral retina. The algorithm was based on a Gaussian model to identify the centers of labeled cells (Wu et al, 2021). The threshold of peak intensity and the variance of distribution were initially determined using visual inspection, and a comparison to the number of manually counted cones from 6 retinas.…”

Augmentation of CD47/SIRPα signaling protects cones in genetic models of retinal degeneration

“…To measure the effect of CD47 on cone survival, GFP-positive cones in the central retinas of rd1 mice were quantified. Cone counting using a GFP vector has been previously validated by our group and provides unambiguous labeling of cones, enabling automated quantification ( 4 , 9 , 10 , 23 ). At the doses used in this study, coinfection with 2 serotype 8 vectors in cones is expected to be at least 90% ( 4 ).…”