Txnip deletions and missense alleles prolong the survival of cones in a retinitis pigmentosa mouse model

Reviewer #1 (Public Review):

Summary:
This is a follow-up study to the authors' previous report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

Strengths:
Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study.

Weaknesses:
I have just one concern that I would like the authors to address. It is about the text that begins at line 133: "We assayed their ability to clear GLUT1 from the RPE surface (Figure 2A)". Please provide more details about this. From the figure it appears that n = 1 for this experiment, but given how careful the authors are with these types of studies that seems unlikely. How did the authors quantify the ability to clear GLUT1 from the surface? Was it cleared from both the apical and basal surface? (It is hard to resolve the apical and basal surfaces in the images provided). The experiments shown in Fig. 1H and Fig. 1I of PMID 31365873 shows how GLUT1 disappears only from the apical surface (under the conditions of that experiment and through the mechanism described in their text). It would be helpful for the authors to discuss their current results in the context of that experiment.

Reviewer #2 (Public Review):

The hard work of the authors is much appreciated. With overexpression of a-arrestin Txnip in RPE, cones and the combined respectively, the authors show a potential gene agnostic treatment that can be applied to retinitis pigmentosa. Furthermore, since Txnip is related to multiple intracellular signaling pathway, this study is of value for research in the mechanism of secondary cone dystrophy as well.

There are a few areas in which the article may be improved through further analysis and application of the data, as well as some adjustments that should be made in to clarify specific points in the article.

Reviewer #3 (Public Review):

Summary:

Xue et al. extended their groundbreaking discovery demonstrating the protective effect of Txnip on cone photoreceptor survival. This was achieved by investigating the protection of cone degeneration through the overexpression of five distinct mutated variants of Txnip within the retinal pigment epithelium (RPE). Moreover, the study explored the roles of two proteins, HSP90AB1 and Arrdc4, which share similarities or associations with Txnip. They found the protection of Txnip in RPE cells and its mechanism is different from its protection in cone cells. These discoveries have significant implications for advancing our understanding of the mechanisms underlying Txnip's protection on cone cells.

Strengths:
1. Identify the roles of different Txnip mutations in RPE and their effects on the expression of glucose transporter
2. Dissect the mechanism of Txnip in RPE vs Cone photoreceptors in retinal degeneration models.
3. Explore the functions of ARrdc4, a protein similar to Txnip and HSP90AB1 in cone degeneration.

Weaknesses:
1. Arrdc4 has deleterious effect on cone survival but no discussion on its mechanism.
2. Inhibition of HSP90 is known to cause retinal generation. It is unclear why inhibition enhances the protection of Txnip.

Author Response

We are pleased that the data presented in our submission was found to be informative and suitable for publication in eLife. The Reviewers made several comments that we address below. They listed three weaknesses of our work: 1) details of RPE GLUT1 immunohistochemistry (IHC), 2) the mechanism of Arrdc4, and 3) the mechanism of HSP90AB1. Additional suggestions made by the Reviewers, aimed at elucidating mechanisms, are of great interest to us, but would require experiments that are beyond the scope of the current work.

We provide the following provisional responses to the identified weaknesses:

1. Reviewer 1 asked several questions regarding the IHC of GLUT1, including the number of retinas examined, the location and quantification of the staining, and our results relative to those of another publication.

We injected more than one eye with each of the AAV-Best1-Txnip alleles.

However, only one of the fully infected eyes of each allele was processed for GLUT1 IHC. We found the GLUT1 removal from the basolateral surface of the RPE by AAV-Best1-Txnip (i.e. the wild type full length allele) was complete, obvious, and consistent from eye to eye, as shown in our original publication (Xue et al., 2021, PMID: 33847261). It was obvious as the GLUT1 on the basolateral surface of the RPE is more easily scored than that on the apical surface. The photoreceptor inner segments and Müller glia microvilli also have GLUT1, and their processes are juxtaposed and/or intertwined with the apical processes of the RPE, making the apical process GLUT1 staining of the RPE much more difficult to score. In some sections where the RPE and the retina separate, we can score the apical process GLUT1 staining of the RPE, but we do not always have this situation in our sections. We should have been more explicit about the location of the IHC signal that we were referring to in the manuscript and will do so in the Revision.

We present images in Figure 2 supplement 1 that are representative for each allele, in the one retina scored for each allele. As Dr. Xue was in the process of moving to China and setting up his own lab at the time of submission, additional retinas were not processed for IHC. However, his laboratory will examine the staining on additional retinas. Given that the results of the wild type allele were very reproducible, we do not anticipate different results from those we have presented for the new alleles. However, the quantification is difficult for the total GLUT1 protein within the RPE due to the ambiguities of staining in the photoreceptors and the Müller glia.

As a separate issue, Reviewer #1 mentioned the work of another group (Wang et al., 2019, PMID: 31365873), which claimed that, on the apical surface of the RPE, GLUT1 is down-regulated in a RP mouse strain, RhoP23H. We have not consistently observed such a down-regulation of GLUT1 in other RP mouse strains such as rd1, rd10 or Rho-/- (unpublished data; see review Xue and Cepko, 2023, PMID: 37460158). However, we would like to point out that it is difficult to score GLUT1 staining on the RPE apical surface, as noted above. It is even more difficult in the degenerating retina where RPE and photoreceptor processes degenerate. For reference, one can see images of degenerating RPE apical processes in Wu et al. 2021 (PMID: 33491671).

1. Little was known about the function of Arrdc4 until very recently. During our submission of this manuscript, a study was published concerning an Arrdc4 global knockout mouse by Richard Lee’s group. They proposed that Arrdc4 is critical for liver glucagon signaling, which could be negated by insulin (Dagdeviren et al, 2023, PMID: 37451484). The implication of this study to RP cone survival is unclear, but interestingly, the activation of insulin/mTORC1 pathway is helpful for RP cone survival, as first discovered by Claudio Punzo when a postdoc in our group (PMID: 19060896, PMID: 25798619).

2. Little is known about the function of HSP90AB1. Recently, Ramamurthy’s group reported that knocking out HSP90AA1, a paralog of HSP90AB1 which has 14% different amino acids, led to rod death and correlated with PDE6 dysregulation (Munezero et al, 2023, PMID: 37172722). However, the exact role of HSP90AA1 in rods needs to be clarified, and the implications for HSP90AB1 in WT and/or RP cones are still unclear.

The above responses will be incorporated to our next version of submission.