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

  1. Yunlu Xue
  2. Yimin Zhou
  3. Constance L. Cepko

  • Curated by eLife

    eLife logo

    eLife assessment

    This fundamental study advances our understanding of the cell specific treatment of cone photoreceptor degeneration by Txnip. The evidence supporting the conclusions is compelling with rigorous genetic manipulation of Txnip mutations. The work will be of broad interest to vision researchers, cell biologists and biochemists.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Retinitis pigmentosa (RP) is a prevalent inherited retinal degenerative disease worldwide, affecting 1 in 4,000 people. The disease is characterized by an initial loss of night vision followed by a loss of daylight and color vision. Many of the RP disease genes are expressed in the rod photoreceptors, the cell type that initiates dim light vision. Following loss of rods, the cone photoreceptors, which initiate daylight vision, also are affected and can die leading to total loss of vision. The reasons for loss of cone vision are not entirely clear, but appear to be due to loss of the rods. Previously we showed that overexpressing Txnip, an α-arrestin protein, in mouse models of RP using AAV gene therapy prolonged the survival of RP cones (Xue et al., 2021). At least part of the mechanism for cone survival was a switch in the fuel source, from glucose to lactate. In addition, the mitochondria of cones were both morphologically and functionally improved by delivery of Txnip. We have gone on to test several alleles of Txnip for the ability to prolong cone survival in rd1 , a mouse model of RP. In addition, proteins that bind to Txnip and/or have homology to Txnip were tested. Five different deletion alleles of Txnip were expressed in cones or the retinal pigmented epithelium (RPE). Here we show that the C-terminal half of Txnip (149-397aa) is sufficient to remove GLUT1 from the RPE cell surface, and improved rd1 cone survival when expressed specifically in the RPE. Overexpressing Arrdc4, an α-arrestin that shares 60% similar protein sequence to Txnip, reduced rd1 cone survival. Reduction of the expression of HSP90AB1, a protein that interacts with Txnip and regulates metabolism, improved the survival of rd1 cones alone and was additive for cone survival when combined with Txnip. However, full length Txnip with a single amino acid change, C247S, as we tested in our original study, remains the most highly efficacious form of the gene for cone rescue. The above observations suggest that only a subset of the hypothesized and known activities of Txnip play a role in promoting RP cone survival, and that the activities of Txnip in the RPE differ from those in cone photoreceptors.

Article activity feed

  1. Version published to 10.7554/elife.90749.3 on eLife
  2. Version published to 10.7554/elife.90749 on eLife
  3. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    This is a follow-up study to the authors' previous eLife 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. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

    We are delighted that Reviewer #1 is satisfied with this revised version.

    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.

    Strengths

    • The follow-up study builds on innovative ground by exploring the impact of TxnipC247S and its combination with HSP90AB1 knockdown on cone survival, offering novel therapeutic pathways.
    • Testing of different Txnip deletion mutants provides a nuanced understanding of its functional domains, contributing valuable insights into the mechanism of action in RP treatment.
    • The findings regarding GLUT1 clearance and the differential effects of Txnip mutants on cone and RPE cells lay the groundwork for targeted gene therapy in RP.

    Weaknesses

    • The focus on specific mutants and overexpression systems might overlook broader implications of Txnip interactions and its variants in the wider context of retinal degeneration.

    Txnip is not expressed in WT or RP cones, as described in our previous study (Xue et al., 2021, eLife), so we could not perform loss of function assays. We thus chose overexpression, and assayed various alleles, based upon the literature, as we describe in our manuscript.

    • The study's reliance on cell count and GLUT1 expression as primary outcomes misses an opportunity to include functional assessments of vision or retinal health, which would strengthen the clinical relevance.

    In our previous study, we demonstrated that the optomotor response of Txnip-treated RP mice improved (Xue et al., 2021, eLife). Also, as described in our previous Txnip study, as well as an independent study (Xue et al., 2021, eLife; Xue et al., 2023, PNAS), ERG assays of Txnip-treated RP cones were no different than the controls. Other therapies that prolong RP cone survival and the optomotor response in our lab also failed to save the ERG, suggesting that there are other pathways that need to be addressed, e.g. the visual cycle. A combination therapy addressing multiple problems is one of our goals.

    • The paper could benefit from a deeper exploration of why certain treatments (like Best1-146 Txnip.C247S) do not lead to cone rescue and the potential for these approaches to exacerbate disease phenotypes through glucose shortages.

    This system is more complicated than we currently understand, and more work needs to be done.

    • Minor inconsistencies, such as the missing space in text references and the need for clarification on data representation (retinas vs. mice), should be addressed for clarity and accuracy.

    The missing spaces are added.

    We described the strategy of injecting the same mouse in each eye, one eye with control and one with the experimental vector. However, the following sentence has been added to the Materials and Methods to better assist the reader:

    “In almost all experiments, other than as noted, one eye of the mouse was treated with control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye), and the other eye was treated with the experimental vector plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye.”

    • The observation of promoter leakage and potential vector tropism issues raise questions about the specificity and efficiency of the gene delivery system, necessitating further discussion and validation.

    The following sentences have been added to the Results. We do not think this phenomenon affects the practice of the experiments or the interpretation of the results in this study.

    “To enable automated cone counting and trace the infection, we co-injected an AAV (AAV8-RedO-H2BGFP-WPRE-bGHpA) encoding an allele of GFP fused to histone 2B (H2BGFP), which localized to the nucleus. As the red opsin promoter was used to express this gene, H2BGFP was seen in cone nuclei, but not in the RPE, if AAV8-RedO-H2BGFP-WPRE-bGHpA was injected alone. However, when an AAV that expressed in the RPE, i.e. AAV8-Best1-Sv40intron-(Gene)-WPRE-bGHpA, was co-injected with AAV8-RedO-H2BGFP-WPRE-bGHpA, H2BGFP was expressed in the RPE, along with expression in cones (Figure 2A). We speculate that this is due to concatenation or recombination of the two genomes, such that the H2BGFP comes under the control of the RPE promoter. This may be due to the high copy number of AAV in the RPE, as it did not happen in the reverse combination, i.e. AAV with an RPE promoter driving GFP and a cone promoter driving another gene, perhaps due to the observation that the AAV genome copy number is »10 fold lower in cones than in the RPE (Wang et al., 2020).”

  4. eLife

    eLife assessment

    This fundamental study advances our understanding of the cell specific treatment of cone photoreceptor degeneration by Txnip. The evidence supporting the conclusions is compelling with rigorous genetic manipulation of Txnip mutations. The work will be of broad interest to vision researchers, cell biologists and biochemists.

  5. eLife

    Reviewer #1 (Public Review):

    Summary:

    This is a follow-up study to the authors' previous eLife 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. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

  6. eLife

    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.

    Strengths

    - The follow-up study builds on innovative ground by exploring the impact of TxnipC247S and its combination with HSP90AB1 knockdown on cone survival, offering novel therapeutic pathways.
    - Testing of different Txnip deletion mutants provides a nuanced understanding of its functional domains, contributing valuable insights into the mechanism of action in RP treatment.
    - The findings regarding GLUT1 clearance and the differential effects of Txnip mutants on cone and RPE cells lay the groundwork for targeted gene therapy in RP.

    Comments on revised version:

    The researchers answered our questions and included additional discussion in the manuscript.

  7. Version published to 10.1101/2023.08.03.551766v3 on bioRxiv
  8. Version published to 10.7554/elife.90749.2 on eLife
  9. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    eLife assessment

    This fundamental study advances our understanding of the cell specific treatment of cone photoreceptor degeneration by Txnip. The evidence supporting the conclusions is convincing with rigorous genetic manipulation of Txnip mutations, however, there are a few areas in which the article may be improved through further analysis and application of the data. The work will be of broad interest to vision researchers, cell biologists and biochemists.

    Reviewer #1 (Public Review):

    Summary:

    This is a follow-up study to the authors' previous eLife 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.

    We repeated all eight AAV-Best1-Txnip alleles for RPE GLUT1 staining with more than three eyes of each condition. We also quantified the GLUT1 intensity on the RPE basal surface. A new Figure 2-figure supplement 1 with these data has been added to this submission. The results and conclusions are similar to those in our initial submission.

    As mentioned in our provisional responses: GLUT1 on the basal 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. The current quantification in the new Figure 2-figure supplement 1 thus concerns only the basal staining.

    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, as we pointed out above, it is difficult to score GLUT1 staining on the RPE apical surface. 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).

    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.

    As mentioned in our provisional responses, little was known about the function of Arrdc4 or HSP90AB1 in cones. We summarize some of the recent discoveries regarding these two proteins in the new Discussion:

    “Arrdc4, the most similar α-arrestin protein to Txnip that also has Arrestin N- and C- domains, accelerated RP cone death when transduced via AAV (Figure 1). This observation suggests that Txnip has unique functions that protect RP cones. Recently, Arrdc4 has been proposed to be critical for liver glucagon signaling, which could be negated by insulin (Dagdeviren et al. 2023). The implication of this potential role in RP cone survival is unclear, but interestingly, the activation of the insulin/mTORC1 pathway is beneficial to RP cone survival (Punzo et al. 2009; Venkatesh et al. 2015).”

    “Little is known about the function of HSP90AB1. Knocking down Hsp90ab1 improved mitochondrial metabolism of skeletal muscle in a diabetic mouse model (Jing et al. 2018). 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). Inhibiting HSP90AA1 with small molecules transiently delayed cone death in human retinal organoids under low glucose conditions (Spirig et al. 2023). However, the exact role of HSP90AA1 in photoreceptors needs to be clarified, and the implications for HSP90AB1 in RP cones are still unclear. ”

    In addition, we used AlphaFold Multimer, an AI algorithm based on AlphaFold-2, to explore the possible interaction between TXNIP, PARP1 and HSP90AB1 in the revision. One of the predicted models is shown as the new Figure 5-figure supplement 2. The C-terminus of Txnip is predicted to link HSP90AB1 and PARP1 together in this model.

    Recommendations for the authors:

    Reviewer #1 (Recommendations For The Authors):

    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.

    See our responses to Review #1’s public review section above.

    Also, is the clearance from the RPE plasma membrane homogenous throughout the RPE monolayer?

    In the area of AAV infection, the effects are very homogenous. In the uninfected area, the clearance does not occur, and we consider the uninfected area of the same eye to be an excellent internal control.

    A statistical analysis (as was provided for other experiments in the manuscript) would help to make the surprising conclusion about C.Txhniip.C247S more convincing.

    In this revision, we used the Mann-Whitney U test with the Bonferroni correction for GLUT1 intensity quantification. For the cone survival statistics, we used the t-test or ANOVA with Dunnett multiple comparison test. The information has been added to each figure legend.

    Another improvement I suggest for this figure is to include normal full length Txnip as a positive control to show how completely it removes GLUT1 from the surface.

    Added. See the new Figure 2-figure supplement 1.

    Another point that should be discussed is - when Txnip prevents GLUT1 from reaching the surface does all the GLUT1 get fully degraded within the cell. A brief description of how Txnip influences GLUT1 stability and localization would be helpful.

    We are unable to track the fate of the GLUT1 after it is removed, i.e. we do not see definitive intracellular staining. We do not know if this is due to degradation or a hidden epitope.

    Minor point

    (1) Confusing citation on lines 99-100: "We previously showed that overexpressing the Txnip wt allele in the RPE using an RPE specific promoter, derived from the Best1 gene (Esumi et al. 2009),.." makes it sound like Esumi et al. is the citation for their previous study, which is not correct.

    We have amended this to: "We previously showed (Xue et al. 2021) that overexpressing the Txnip wt allele in the RPE using an RPE-specific promoter, derived from the Best1 gene (Esumi et al., 2009), did not improve RP cone survival."

    Reviewer #2 (Recommendations For The Authors):

    Regarding the manuscript, here are some suggestions that authors can take into consideration for the completeness of the study:

    (1) The text references the relationship between α-arrestin and glucose metabolism in cone cells, but fails to provide an explanation for its specific involvement in glucose metabolism. Consequently, readers may struggle to discern the targeted metabolic pathway.

    We understand this point from Reviewer, and would love to know more about its mechanism, which is one reason why we undertook the current study. The mechanism(s) by which Txnip affects metabolism remains to be elucidated. To summarize our findings from our previous study, we showed that LDHB, which converts lactate to pyruvate, was required for Txnip-mediated rescue. Addition of the LDHB gene, however, did not boost rescue. We also showed that mitochondrial size and membrane potential were improved, and the Na/K pump function was improved, in Txnip-treated cones. Improved mitochondria were not sufficient, however, as revealed by a PARP-1 KO mouse with improved mitochondria that did not extend cone survival. In addition, using a Txnip mutant that does not remove the glucose transporter, we still saw cone rescue, so this function cannot be required for Txnip-mediated rescue. How does Txnip lead to improved mitochondria and to a reliance on lactate? We do not know.

    (2) Although the author conducted an experiment on arrdc14 due to its similarity to Txnip, the lack of clarification on why arrdc4, with a 60% amino acid similarity, did not yield the same effects as Txnip remains unaddressed. Highlighting structural disparities or differences in intracellular signaling pathways could potentially shed light on this incongruity. Subsequently, an additional experiment may be warranted to test the hypothesis regarding the effective component of α-arrestin for cone rescue.

    Additional experiments are needed to learn of the relevant differences between Arrdc4 and Txnip, but are beyond the scope of our work at the present. However, we have added a paragraph on newly published data on the function of Arrdc4 in the new Discussion:

    “Arrdc4, the most similar α-arrestin protein to Txnip that also has Arrestin N- and C- domains, accelerated RP cone death when transduced by AAV (Figure 1). This observation suggests that Txnip has unique functions that protect RP cones. Recently, Arrdc4 has been proposed to be critical for liver glucagon signaling, which could be negated by insulin (Dagdeviren et al. 2023). The implication of this potential role regarding RP cone survival is unclear, but interestingly, the activation of the insulin/mTORC1 pathway is beneficial to RP cone survival (Punzo et al. 2009; Venkatesh et al. 2015).”

    (3) The utilization of distinct mutant Txnip variants to impact RPE, cones, and their combined influence is noted. A comparative table elucidating the impact of cone rescue on these three targets would greatly enhance clarity.

    We presented these data in Figure 4 in a table format.

    Additionally, the text does not definitively establish whether Txnip.C247S.LL351 and 352AA, as well as Txnip.C247S, indeed manifest discrepancies when exclusively affecting RPE.

    We edited a sentence in Results to: “Similar to Best1-wt Txnip (Xue et al., 2021), Best1-Txnip.C247S did not show significant improvement of cone survival, ruling out the C247S mutation alone as promoting the cone survival by Best1-Txnip.C247S.LL351 and 352AA.”

    (4) While the text mentions that Txnip stimulates lactate utilization within cones, it remains unclear whether this effect extends to RPE. If applicable, this trait could potentially contribute to its role in cone rescue.

    We agree with the Reviewer, and hope to address this question in our next study.

    (5) The discussion introduces the notion that one potential mechanism for cone rescue by Txnip.C247S involves facilitating unhindered movement of Thioredoxin for redox processes. To validate this hypothesis and elucidate the mechanics of Txnip's involvement in cone rescue, it may be prudent to conduct further experiments concentrating on the interaction between Txnip and thioredoxin. Alternatively, an experiment aimed at upregulating Thioredoxin expression would be a valuable addition.

    We hope to address this question in the future. However, the effect may be more complicated than our simple hypothesis regarding release of Thioredoxin. More than a dozen proteins were found to differentially interact with Txnip vs. Txnip.C247S (Forred et al. 2016).

    Reviewer #3 (Recommendations For The Authors):

    (1) Glucose transporter 1 is identified as an important mechanism in the protection of cone degeneration. It is unclear why GLut1 is upregulated in retinal cells although the expression of Txnip mutants are specifically in the RPE in Figure 2.

    This retinal GLUT1 upregulation was not consistently observed in the treated eyes, so we did not comment on it in the text.

    (2) Mutant N. Txnip was mentioned in the discussion that it causes obvious retinal degeneration. The quantification of retinal thickness from Figure 2 will be more rigorous.

    Unlike the robust effects of Best1-N.Txnip on RPE GLUT1 level, this negative effect of Best1-N.Txnip on ONL thickness was not consistent. This result does not undermine the other major conclusions. Therefore, we deleted the related sentence of the original text: “This hypothesis is supported by the observation that N.Txnip led to an obvious thinning of the outer nuclear layer of the wt retina, reflecting a loss of photoreceptors”. We did leave in the related finding as follows:

    “The N-terminal half of Txnip (1-228aa) might exert harmful effects in the RPE, that negate the beneficial effects from the C-terminal half, suggested by the observation that its removal, in the C-terminal 149-397 allele, led to better cone survival when expressed in the RPE (Figure 2). In cones, the C-terminal half, including the C-terminal IDR tail, may cooperate with the N-terminal half, or negate its negative effects, to benefit RP cone survival. However, the C-terminal half is not sufficient for cone rescue when expressed in cones, as the 149-397 allele did not rescue.”

  10. eLife

    eLife assessment

    This fundamental study advances our understanding of the cell specific treatment of cone photoreceptor degeneration by Txnip. The evidence supporting the conclusions is compelling with rigorous genetic manipulation of Txnip mutations. The work will be of broad interest to vision researchers, cell biologists and biochemists.

  11. eLife

    Reviewer #1 (Public Review):

    Summary:

    This is a follow-up study to the authors' previous eLife 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. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

  12. eLife

    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.

    Strengths

    - The follow-up study builds on innovative ground by exploring the impact of TxnipC247S and its combination with HSP90AB1 knockdown on cone survival, offering novel therapeutic pathways.
    - Testing of different Txnip deletion mutants provides a nuanced understanding of its functional domains, contributing valuable insights into the mechanism of action in RP treatment.
    - The findings regarding GLUT1 clearance and the differential effects of Txnip mutants on cone and RPE cells lay the groundwork for targeted gene therapy in RP.

    Weaknesses

    - The focus on specific mutants and overexpression systems might overlook broader implications of Txnip interactions and its variants in the wider context of retinal degeneration.
    - The study's reliance on cell count and GLUT1 expression as primary outcomes misses an opportunity to include functional assessments of vision or retinal health, which would strengthen the clinical relevance.
    - The paper could benefit from a deeper exploration of why certain treatments (like Best1-146 Txnip.C247S) do not lead to cone rescue and the potential for these approaches to exacerbate disease phenotypes through glucose shortages.
    - Minor inconsistencies, such as the missing space in text references and the need for clarification on data representation (retinas vs. mice), should be addressed for clarity and accuracy.
    - The observation of promoter leakage and potential vector tropism issues raise questions about the specificity and efficiency of the gene delivery system, necessitating further discussion and validation.

  13. Version published to 10.1101/2023.08.03.551766v2 on bioRxiv
  14. Version published to 10.7554/elife.90749.1 on eLife
  15. eLife

    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.

  16. eLife

    eLife assessment

    This fundamental study advances our understanding of the cell specific treatment of cone photoreceptor degeneration by Txnip. The evidence supporting the conclusions is convincing with rigorous genetic manipulation of Txnip mutations, however, there are a few areas in which the article may be improved through further analysis and application of the data. The work will be of broad interest to vision researchers, cell biologists and biochemists.

  17. eLife

    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.

  18. eLife

    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.

  19. eLife

    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.

  20. Version published to 10.1101/2023.08.03.551766v1 on bioRxiv