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

  1. Yunlu Xue
  2. Sean K Wang
  3. Parimal Rana
  4. Emma R West
  5. Christin M Hong
  6. Helian Feng
  7. David M Wu
  8. Constance L Cepko  Is a corresponding author
  1. Department of Genetics, Blavatnik Institute, Harvard Medical School, United States
  2. Department of Ophthalmology, Harvard Medical School, United States
  3. Howard Hughs Medical Institute, United States
  4. Department of Biostatistics, Harvard T.H. Chan School of Public Health, United States
  5. Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, United States
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Cite this article as: eLife 2021;10:e66240 doi: 10.7554/eLife.66240

Abstract

Retinitis pigmentosa (RP) is an inherited retinal disease affecting >20 million people worldwide. Loss of daylight vision typically occurs due to the dysfunction/loss of cone photoreceptors, the cell type that initiates our color and high-acuity vision. Currently, there is no effective treatment for RP, other than gene therapy for a limited number of specific disease genes. To develop a disease gene-agnostic therapy, we screened 20 genes for their ability to prolong cone photoreceptor survival in vivo. Here, we report an adeno-associated virus vector expressing Txnip, which prolongs the survival of cone photoreceptors and improves visual acuity in RP mouse models. A Txnip allele, C247S, which blocks the association of Txnip with thioredoxin, provides an even greater benefit. Additionally, the rescue effect of Txnip depends on lactate dehydrogenase b (Ldhb) and correlates with the presence of healthier mitochondria, suggesting that Txnip saves RP cones by enhancing their lactate catabolism.

eLife digest

Retinitis pigmentosa is an inherited eye disease affecting around one in every 4,000 people. It results from genetic defects in light sensitive cells of the retina, called photoreceptor cells, which line the back of the eye. Though vision loss can occur from birth, retinitis pigmentosa usually involves a gradual loss of vision, sometimes leading to blindness. Rod photoreceptors, which are responsible for vision in low light, are impacted first. The disease then affects cone photoreceptors, the cells that detect light during the day, providing both color and sharp vision.

Around 100 mutated genes associated with retinitis pigmentosa have been identified, but only a handful of families with one of these mutant genes have been treated with a gene therapy specific for their mutated gene. There are currently no therapies available to treat the vast number of people with this disease. The mutations that cause retinitis pigmentosa directly affect the rod cells that detect dim light, leading to loss of night vision. There is also an indirect effect that causes cone photoreceptors to stop working and die. One theory to explain this two-step disease process relates to the fact that cone photoreceptors are very active cells, requiring a high level of energy, nutrients and oxygen. If surrounding rod cells die, cone photoreceptors may be deprived of some essential supplies, leading to cone cell death and daylight vision loss.

To examine this theory, Xue et al. tested a new gene therapy designed to alleviate the potential shortfall in nutrients. The experiments used three different strains of mice that had the same genetic mutations as humans with retinitis pigmentosa. The gene therapy used a virus, called adeno-associated virus (AAV), to deliver 20 different genes to cone cells. Each of the 20 genes tested plays a different role in cells’ processing of nutrients to provide energy. After administering the treatment, Xue et al. monitored the mice to see whether or not their vision was affected, and how cone cells responded.

Only one of the 20 genes, Txnip, delivered using gene therapy, had a beneficial effect, prolonging cone cell survival in all three mouse strains. The mice that received Txnip also retained their ability to discern moving stripes on vision tests. Further investigations demonstrated that activating Txnip forced the cones to start using a molecule called lactate as an energy source, which could be more available to them than glucose, their usual fuel. These cells also had healthier mitochondria – the compartments inside cells that produce and manage energy supplies. This dual effect on fuel use and mitochondrial health is thought to be the basis for the extended cone survival and function.

These experiments by Xue et al. have identified a good gene therapy candidate for treating retinitis pigmentosa independently of which genes are causing the disease. Further research will be required to test the safety of the gene therapy, and whether its beneficial effects translate to humans with retinitis pigmentosa, and potentially other diseases with unhealthy photoreceptors.

Introduction

Retinitis pigmentosa (RP) is one of the most prevalent types of inherited retinal diseases affecting approximately 1 in ~4,000 people (Hartong et al., 2006). In RP, the rod photoreceptors, which initiate night vision, are primarily affected by the disease genes and degenerate first. The degeneration of cones, the photoreceptors that initiate daylight, color, and high-acuity vision, then follows, which greatly impacts the quality of life. Currently, one therapy that holds great promise for RP is gene therapy using adeno-associated virus (AAV) (Maguire et al., 2019). This approach has proven successful for a small number of genes affecting a few disease families (Cehajic-Kapetanovic et al., 2020). However, due to the number and functional heterogeneity of RP disease genes (≈100 genes that primarily affect rods, https://sph.uth.edu/retnet/), gene therapy for each RP gene will be logistically and financially difficult. In addition, a considerable number of RP patients do not have an identified disease gene. A disease gene-agnostic treatment aimed at prolonging cone function/survival in the majority of RP patients could thus benefit many more patients. Given that the disease gene is typically not expressed in cones, and thus their death is due to non-autonomous mechanisms that may be in common across affected families, answers to the question of why cones die may provide an avenue to a widely applicable therapy for RP. To date, the suggested mechanisms of cone death include oxidative damage (Komeima et al., 2006; Wellard et al., 2005; Xiong et al., 2015), inflammation (Wang et al., 2020; Wang et al., 2019; Zhao et al., 2015), and a shortage of nutrients (Aït-Ali et al., 2015; Kanow et al., 2017; Punzo et al., 2012; Punzo et al., 2009; Wang et al., 2016).

In 2009, we surveyed gene expression changes that occurred during retinal degeneration in four mouse models of RP (Punzo et al., 2009). Those data led us to suggest a model wherein cones starve and die due to a shortage of glucose, which is typically used for energy and anabolic needs in photoreceptors via glycolysis. Evidence of this ‘glucose shortage hypothesis’ was subsequently provided by orthogonal approaches from other groups (Aït-Ali et al., 2015; Wang et al., 2016). These studies have inspired us to test 20 genes that might affect the uptake and/or utilization of glucose by cones in vivo in three mouse models of RP (Figure 1—source data 1). Only one gene, Txnip, had a beneficial effect, prolonging cone survival and visual acuity in these models. Txnip encodes an α-arrestin family member protein with multiple functions, including binding to thioredoxin (Junn et al., 2000; Nishiyama et al., 1999), facilitating removal of the glucose transporter 1 (GLUT1) from the cell membrane (Wu et al., 2013), and promoting the use of non-glucose fuels (DeBalsi et al., 2014). Because α-arrestins are structurally distinct from the visual or β-arrestins, such as ARR3, Txnip is unlikely to bind to opsins or to participate in phototransduction (Hwang et al., 2014; Kang et al., 2015; Puca and Brou, 2014). We tested a number of Txnip alleles and found that one allele, C247S, which blocks the association of Txnip with thioredoxin (Patwari et al., 2006), provided the greatest benefit. Investigation of the mechanism of Txnip rescue revealed that it required lactate dehydrogenase b (Ldhb), which catalyzes the conversion of lactate to pyruvate. Imaging of metabolic reporters demonstrated an enhanced intracellular ATP:ADP ratio when the retina was placed in lactate medium. Moreover, by several measures, mitochondria appeared to be healthier as a result of Txnip addition, but this improvement was not sufficient for cone rescue.

The above observations led to a model wherein Txnip shifts cones from their normal reliance on glucose to enhanced utilization of lactate, as well as marked improvement in mitochondrial structure and function. Analysis of the rescue activity of several additional genes predicted to affect glycolysis provided support for this model. Finally, as our goal is to rescue cones that suffer not only from metabolic challenges, but also from inflammation and oxidative damage, we tested Txnip in combination with anti-inflammatory and anti-oxidative damage genes, and found additive benefits for cones. These treatments may benefit cones not only in RP, but also in other ocular diseases where similar environmental stresses are present, such as in age-related macular degeneration (AMD).

Results

Txnip prolongs RP cone survival and visual acuity

We delivered genes that might address a glucose shortage and/or mismanagement of metabolism in a potentially glucose-limited environment. To this end, 12 AAV vectors were constructed to test genes singly or in combination for an initial screen (Figure 1—figure supplement 1E). Subsequently, an additional set of AAV vectors were made based upon the initial screen results, as well as other rationales, to total 20 genes tested in all (Figure 1—source data 1). Most of these vectors carried genes to augment the utilization of glucose, such as hexokinases, phosphofructokinase, and pyruvate kinase. Each AAV vector used a cone-specific promoter, which was previously found to be non-toxic at the doses used in this study (Xiong et al., 2019). An initial screen was carried out in rd1 mice, which harbor a null allele in the rod-specific gene, Pde6b. This strain has a rapid loss of rods, followed by cone death. The vectors were subretinally injected into the eyes of neonatal rd1 mice, in combination with a vector using the human red opsin (RedO) promoter, to express a histone 2B-GFP fusion protein (AAV-RedO-H2BGFP). The H2BGFP provides a very bright cone-specific nuclear labeling, enabling automated quantification. As a control, eyes were injected with AAV-RedO-H2BGFP alone. Rd1 cones begin to die at ≈postnatal day 20 (P20) after almost all rods have died (Figure 1—figure supplement 1A, Figure 1—figure supplement 2A). The number of rd1 cones was quantified by counting the H2BGFP+ cells using a custom-made MATLAB program (Figure 1A, Figure 1—figure supplement 1C). Because ~11,000 rd1 cones were counted in the central ½ radius of retina before their death at P20 (Figure 1—figure supplement 1E), we estimated ~20% H2BGFP labeling efficiency using data for wildtype mice for comparison (i.e., ~50,000 cones within ½ radius of wildtype retina) (Jeon et al., 1998), with this injection dose. Only cones within the central ½ radius region of the retina were counted since RP cones in the periphery die much later (Hartong et al., 2006; Punzo et al., 2009). Among the vectors with individual or combinations of genes, only Txnip preserved rd1 cones at P50 (Figure 1A, B, Figure 1—figure supplement 1E). The effects were likely on cone survival as it did not change the number of cones at P20 prior to their death, but did provide survival benefit by ~P30 (Figure 1A, B, Figure 1—figure supplement 2A–C). The level of Txnip rescue in P50 rd1 cones was comparable to that seen using AAV with a cytomegalovirus (CMV) promoter to express a transcription factor, Nrf2, that regulates anti-oxidation pathways and reduces inflammation, as we found previously (Xiong et al., 2015Figure 1—figure supplement 1E). One combination led to a reduction in cone survival, that of Hk1 plus Pfkm (Figure 1—figure supplement 1E).

Figure 1 with 2 supplements see all
Txnip effects on cone survival and cone-mediated vision in retinitis pigmentosa (RP) mice.

(A) Representative images from postnatal day 20 (P20) and P50 rd1, P130 rd10, and P150 Rho-/- flat-mounted retinas, in which retinas were infected with adeno-associated viruses (AAVs) encoding Txnip and H2BGFP (AAV8-RedO-Txnip, ≈1 × 109 vg/eye plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye) or control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). The outer circle was drawn to mark the outline of the retina, and the inner circle was drawn to the ½ radius of the outer circle. The small boxes in the top four panels mark the regions shown at higher magnification in Figure 1—figure supplement 1C, demonstrating the pixels recognized as cones by the MATLAB automated-counting program. The number at the lower-right corner in each panel is the count of cones within the ½ radius of each image. All H2BGFP-labeled cones were counted within the central retina defined by the ½ radius (i.e., not just the cells from the small boxes). (B) Quantification of H2BGFP-positive cones within the ½ radius of the retina for different groups (same as in A). Error bar: standard deviation. The number in the round brackets ‘()’ indicates the sample size, that is, the number of retinas within each group. (C) Visual acuity of rd10 and Rho-/- mice transduced with Txnip or H2BGFP alone in each eye measured using an optomotor assay. Error bar: SEM. NS: not significant; p>0.05, *p<0.05, **p<0.01, ***p<0.001, **** p< or <<0.0001. RedO: red opsin promoter; AAV: adeno-associated virus.

Figure 1—source data 1

Adeno-associated virus 8 vectors used in this study.

Best1: retinal pigmented epithelium-specific promoter; SynPVI, SynP136, red opsin (RedO), RO1.7: various cone-specific promoters; N/A: not applicable; –: not performed; Pos: positive for cone rescue; Neg: negative for cone rescue.

https://cdn.elifesciences.org/articles/66240/elife-66240-fig1-data1-v2.docx

Our initial screen used the RedO promoter to drive Txnip expression. To evaluate a different cone-specific promoter, Txnip also was tested using a newly described cone-specific promoter, SynPVI (Jüttner et al., 2019). This promoter also led to prolonged cone survival (Figure 1—figure supplement 1E). To explore whether Txnip gene therapy is effective beyond rd1, it was tested in rd10 mice, which carry a missense Pde6b mutation, and in Rho-/- mice, which carry a null allele in a rod-specific gene, rhodopsin. Cone survival was evaluated after the majority of central cones had died, with different ages for different strains, based upon our previous work (Punzo et al., 2009; Wang et al., 2019; Xiong et al., 2015). Both rd10 and Rho-/- mice showed improved cone survival (Figure 1A, B). The rescue effect did not persist long term, however, as by P240 in the Rho-/- strain it was not significant (Figure 1—figure supplement 2D). To determine if Txnip-transduced mice sustained greater visual performance than control RP mice, an optomotor assay was used to measure maximal visual threshold for spatial frequency (i.e., visual acuity) (Prusky et al., 2004). Under conditions that simulated daylight, Txnip-transduced eyes showed enhanced visual acuity compared to the control contralateral eyes in rd10 and Rho-/- mice (Figure 1C). The rd1 strain degenerates so quickly that it could not be evaluated in this assay. To determine if there was an improvement in overall cone phototransduction, summed across all cones, electroretinography (ERG) was carried out. No effect was observed in rd10 mice transduced with Txnip (Figure 1—figure supplement 2E). Txnip also was evaluated for effects on cones in wildtype (WT) mice using peanut agglutinin (PNA) staining, which stains the cone-specific extracellular matrix and reflects cone health. No effect was seen on PNA staining (Figure 1—figure supplement 1D). In addition, retinas from both WT and P21 rd1 mice were stained using anti-ARR3, which stains the entire cone. At P31, the approximate number and morphology of Txnip-transduced cones in WT retinas was similar to uninfected WT retinas (Figure 1—figure supplement 2A). At P21 and P30, immunohistochemistry (IHC) for ARR3 in rd1 retinas did not show an obvious rescue of cone outer segments by Txnip (Figure 1—figure supplement 2A).

Evaluation of Txnip alleles for cone survival

Previous studies of Txnip provided a number of alleles that could potentially lead to a more effective cone rescue by Txnip and/or provide some insight into which of the Txnip functions are required for enhancing cone survival. A C247S mutation has been shown to block Txnip’s inhibitory interaction with thioredoxin (Patwari et al., 2009; Patwari et al., 2006), which is an important component of a cell's ability to fight oxidative damage via thiol groups (Junn et al., 2000; Nishinaka et al., 2001; Nishiyama et al., 1999). If cone rescue by Txnip required this function, the C247S allele should be less potent for cone rescue. Alternatively, if loss of thioredoxin binding freed Txnip for its other functions and made more thioredoxin available for oxidative damage control, this allele might more effectively promote cone survival. The C247S clearly provided more robust cone rescue than WT Txnip in all three RP mouse strains (Figure 2, Figure 2—figure supplement 1A, B). 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. The S308A change did not benefit cone survival in either context (Figure 2). In addition, the S308A allele was assayed for negative effects on cones by an assessment of rd1 cone number prior to P20, that is, before the onset of cone death (Figure 2—figure supplement 1C). It did not reduce the cone number at this early timepoint, indicating that Txnip.S308A was not toxic to cones. This finding suggests that the S308 residue is critical for the therapeutic function of Txnip through an unclear mechanism. One additional set of amino acid changes, LL351 and 352AA, was tested in the context of C247S. This allele eliminates a clathrin-binding site, and thus hampers Txnip’s ability to remove GLUT1 from cell surface through clathrin-coated pits (Wu et al., 2013). Txnip.C247S.LL351 and 352AA could still delay RP cone death compared to the control (Figure 2B), suggesting that the therapeutic effect of Txnip was unlikely to be only through the removal of GLUT1 from the cell surface. To further explore the role of GLUT1, an shRNA to Slc2a1, which encodes GLUT1, was tested. It did not prolong RP cone survival (Figure 2—figure supplement 1D). The slight decrease of Txnip.C247S.LL351 and 352AA in cone rescue compared to Txnip.C247S might be due to other, currently unknown effects of LL351 and 352, or a less specific effect, for example, a protein conformational change.

Figure 2 with 2 supplements see all
Test of Txnip alleles on cone survival.

(A) Representative P50 rd1 flat-mounted retinas after P0 infection with one of five different Txnip alleles (AAV8-RedO-Txnip wildtype (WT)/.C247S/.S308A/.C247S.S308A/.C247S.LL351 and 352AA, ≈1 × 109 vg/eye, plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye), or control eyes infected with AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye alone. (B) Quantification of H2BGFP-positive cones within the ½ radius of P50 rd1 retinas transduced with WT Txnip, Txnip alleles, and control (same as in A). The number in the round brackets ‘()’ indicates the sample size, that is, the number of retinas within each group. Error bar: standard deviation. Txnip.CS.SA: Txnip.C247S.S308A; Txnip.CS.LLAA: Txnip.C247S.LL351 and 352AA. NS: not significant, p>0.05, *p<0.05, **p<0.01, ***p<0.001, **** p< or <<0.0001. RedO: red opsin promoter; AAV: adeno-associated virus.

Txnip requires Ldhb to prolong cone survival

People with Txnip null mutations present with lactic acidosis (Katsu-Jiménez et al., 2019), suggesting that Txnip deficiency might compromise lactate catabolism. A metabolomic study of muscle using a targeted knockout of Txnip suggested that Txnip increases the catabolism of non-glucose fuels, such as lactate, ketone bodies, and lipids (DeBalsi et al., 2014). This switch in fuel preference was proposed to benefit the mitochondrial tricarboxylic acid cycle (TCA cycle), leading to a greater production of ATP. As presented earlier, a problem for cones in the RP environment might be a shortage of glucose (Aït-Ali et al., 2015; Punzo et al., 2009; Wang et al., 2016). A benefit of Txnip might then be to enable and/or force cells to switch from a preference for glucose to one or more alternative fuels. To test this hypothesis, we co-injected AAV-Txnip with shRNAs targeting the mRNAs for the rate-limiting enzymes for the catalysis of lactate, ketones, or lipids. Ldhb, encoded by the Ldhb gene, is the enzyme that converts lactate to pyruvate to potentially fuel the TCA cycle, and lactate dehydrogenase a (Ldha, encoded by Ldha gene) converts pyruvate to lactate (Eventoff et al., 1977). We found that Txnip rescue was significantly decreased by any one of three Ldhb shRNAs (siLdhb) or by overexpression of Ldha (Figure 3A, B, Figure 3—figure supplement 1B–E). We also tested the rescue effect of Txnip plus an shRNA against Oxct1 (siOxct1), a critical enzyme for ketolysis (Zhang and Xie, 2017), or against Cpt1a (siCpt1a), a component for lipid transporter that is rate limiting for β-oxidation (Shriver and Manchester, 2011). These shRNAs, tested singly or in combination, did not reduce the effectiveness of Txnip rescue (Figure 3C). Taken together, these data support the use of lactate, but not ketones or lipids, as a critical alternative fuel for cones when Txnip is overexpressed.

Figure 3 with 4 supplements see all
Effect of knockdown of lactate dehydrogenase b (Ldhb) in Txnip-transduced retinitis pigmentosa cones in vivo.

(A) Representative P50 rd1 flat-mounted retinas after P0 infection with control shRNA construct (siNC) or an shRNA construct targeting Ldhb (siLdhb(#2)) in the presence or absence of transduced Txnip (AAV8-RedO-Txnip, ≈1 × 109 vg/eye; AAV8-RedO-shRNA ≈1 × 109 vg/eye), plus AAV8-RedO-H2BGFP (2.5 × 108 vg/eye) (B) Quantification of H2BGFP-positive cones within the ½ radius of P50 rd1 retinas transduced with control, siNC control, Txnip + siLdhb(#2), or Txnip + siNC control (same as in A). (C) Quantification of H2BGFP-positive cones within the ½ radius of P50 rd1 retinas transduced with Txnip + siOxct1(#c), Txnip + siCpt1a(#c), Txnip + siOxct1(#c) + siCpt1a(#c), or siNC control. (All are AAV8-RedO-Txnip, ≈1 × 109 vg/eye; AAV8-RedO-shRNA, ≈1 × 109 vg/eye; plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye.) Error bar: standard deviation. NS: not significant, p>0.05, **p<0.01, ***p<0.001, **** p< or <<0.0001. RedO: red opsin promoter; AAV: adeno-associated virus.

Txnip improves the ATP:ADP ratio in RP cones in the presence of lactate

If the improved survival of cones following Txnip overexpression is due to improved utilization of non-glucose fuels, cones might show improved mitochondrial metabolism. To begin to examine the metabolism of cones, we first attempted to perform metabolomics of cones with and without Txnip. However, so few cones are present in these retinas that we were unable to achieve reproducible results. An alternative assay was conducted to measure the ratio of ATP to ADP using a genetically encoded fluorescent sensor (GEFS). AAV was used to deliver PercevalHR, an ATP:ADP GEFS (Tantama et al., 2013), to rd1 cones with and without AAV-Txnip. The infected P20 rd1 retinas were explanted and imaged in three different types of media to measure the cone intracellular ratio of ATP:ADP. Txnip increased the ATP:ADP ratio (i.e., higher FPercevalHR488:405) of rd1 cones in lactate-only medium. This was also seen in pyruvate-only medium, perhaps due to improved mitochondrial health (i.e., greater oxidative phosphorylation [OXPHOS] activity). Consistent with the role of Txnip in removing GLUT1 from the plasma membrane, Txnip-transduced cones had a lower ATP:ADP ratio (i.e., lower FPercevalHR488:405) in high-glucose medium (Figure 4A, B). To further probe whether intracellular glucose was reduced after overexpression of Txnip (Wu et al., 2013), a glucose sensor iGlucoSnFR was used (Keller et al., 2019). This sensor showed reduced intracellular glucose in Txnip-transduced cones (Figure 4—figure supplement 1A, B). Because the fluorescence of GEFS may also be subject to environmental pH, we used a pH sensor, pHRed (Tantama et al., 2011), to determine if the changes of PercevalHR and iGlucoseSnFR were due to a change in pH, and found no significant pH change (Figure 4—figure supplement 1C, D). We also found that lactate, but not pyruvate, utilization by Txnip-transduced cones was critically dependent upon Ldhb for ATP production as introduction of siLdhb abrogated the increase in ATP:ADP in Txnip-transduced cones (Figure 4C). Furthermore, in correlation with improved cone survival by Txnip.C247S compared to WT Txnip (Figure 2B), cones had a higher ATP:ADP ratio in lactate medium when Txnip.C247S was used relative to WT Txnip (Figure 4D, E). Similarly, in correlation with no survival benefit when transduced with Txnip.S308A (Figure 2B), there was no difference in the ATP:ADP ratio when Txnip.S308A was used, relative to control, in lactate medium (Figure 4D, E).

Figure 4 with 1 supplement see all
Effect of Txnip on ATP:ADP levels in retinitis pigmentosa (RP) cones in media with different carbon sources.

(A) Representative ex vivo live images of PercevalHR-labeled cones in P20 rd1 retinas cultured with high-glucose, lactate-only, or pyruvate-only medium and transduced with Txnip (AAV8-RedO-Txnip, 1 × 109 vg/eye, plus AAV8-RO1.7-PercevalHR, 1 × 109 vg/eye) (RO1.7 is a shorter version of the red opsin [RedO] promoter with a similar expression pattern) or control (i.e., AAV8-RO1.7-PercevalHR, 1 × 109 vg/eye). Magenta: fluorescence by 405 nm excitation, indicating low-ATP:ADP; green: fluorescence by 488 nm excitation, indicating high-ATP:ADP. (B) Quantification of normalized PercevalHR fluorescence intensity ratio (FPercevalHRex488nm: ex405nm, proportional to ATP:ADP ratio) in cones from P20 rd1 retinas in different conditions. The number in the square brackets ‘[]’ indicates the sample size, that is, the number of images taken from regions of interest of multiple retinas (≈3 images per retina), in each condition. (C) Quantification of normalized PercevalHR fluorescence intensity of retinas infected with Txnip + siLdhb(#2) and Txnip + siNC in cones from P20 rd1 retina in lactate-only or pyruvate-only medium. (AAV8-RedO-Txnip, ≈1 × 109 vg/eye; AAV8-RedO-shRNA ≈1 × 109 vg/eye; plus AAV8-RO1.7-PercevalHR, 1 × 109 vg/eye.) (D) Representative ex vivo live images of PercevalHR-labeled cones in P20 rd1 retinas cultured in lactate-only medium, following transduction with Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 × 109 vg/eye) or Txnip.S308A (AAV8-RedO-Txnip.S308A, 1 × 109 vg/eye). Magenta: fluorescence by 405 nm excitation, indicating low-ATP:ADP; green: fluorescence by 488 nm excitation, indicating high-ATP:ADP. (E) Quantification of normalized PercevalHR fluorescence intensity following transduction by Txnip, Txnip alleles, and control cones in P20 rd1 retinas cultured in lactate-only medium. Error bar: standard deviation. NS: not significant, p>0.05, **p<0.01, ***p<0.001, **** p< or <<0.0001. AAV: adeno-associated virus.

Txnip improves RP cone mitochondrial gene expression, size, and function

To further probe the mechanism(s) of Txnip rescue, we first tested if all of the benefits of Txnip were due to Txnip's effects on Ldhb. Ldhb was thus overexpressed alone or with Txnip. Ldhb alone did not prolong cone survival, nor did it increase the Txnip rescue (Figure 8—figure supplement 1D). An additional experiment was carried out to investigate if there might be a shortage of the mitochondrial pyruvate carrier, which could limit the uptake of pyruvate into the mitochondria of photoreceptors for ATP synthesis (Grenell et al., 2019). The pyruvate carrier, which is a dimer encoded by Mpc1 and Mpc2 genes, was overexpressed, but did not prolong rd1 cone survival (Figure 7—figure supplement 1C). To take a less biased approach, the transcriptomic differences between Txnip-transduced and control RP cones were characterized. H2BGFP-labeled RP cones were isolated by FACS sorting at an age when cones were beginning to die, and RNA sequencing was performed (Figure 5—figure supplement 1A). Data were obtained from two RP strains, rd1 and Rho-/-. By comparing the differentially expressed genes in common between the two strains, relative to control, 7 genes were seen to be upregulated and 17 were downregulated (Figure 5—source data 1). Three of the seven upregulated genes were mitochondrial electron transport chain (ETC) genes. The upregulation of these three ETC genes in Txnip-transduced rd1 cones was confirmed by ddPCR (Figure 5—figure supplement 1B). Similarly, we also looked for transcriptomic differences induced by Txnip in WT cones using C57BL/6J and BALB/c mice, and found only Txnip mRNA upregulation in common (Figure 5—figure supplement 2A, Figure 5—source data 2). Interestingly, there was almost no Txnip mRNA detected by RNA-seq in the WT or RP control cones, but there was high number of Txnip transcripts following addition of RedO-Txnip in all strains (Figure 5—source data 3).

The finding of upregulated ETC genes in Txnip-transduced RP cones, but not in WT cones, suggested effects of Txnip on mitochondria during cone degeneration. Murakami et al. previously showed that cone mitochondria were swollen and deteriorated in rd10 mouse retinas (Murakami et al., 2012). The morphology of Txnip-transduced mitochondria in RP cones and uninjected WT cones was examined by transmission electron microscopy (TEM) (Figure 5—figure supplement 2B–D). There was an increase in the size of mitochondrial cross sections by Txnip transduction, with a greater increase following transduction with Txnip.C247S in P20 rd1 cones (Figure 5A, B). Mitochondrial membrane potential (ΔΨm), a reflection of mitochondrial ETC function, was also examined using JC-1 dye staining of freshly explanted Txnip-transduced P20 rd1 retinas (Reers et al., 1995). Both Txnip and Txnip.C247S increased the ratio of J-aggregates:JC1-monomers (Figure 5C, D), indicating an increased ΔΨm and/or a greater number/size of mitochondria with a high ΔΨm following Txnip overexpression. This finding was further investigated in vivo using infection by an AAV encoding mitoRFP, which only accumulates in mitochondria with a high ΔΨm (Brodier et al., 2020; Hood et al., 2003). Compared to the control cones without Txnip transduction, the intensity of mitoRFP was higher in P20 rd1 cones transduced with Txnip (Figure 5—figure supplement 1C, D).

Figure 5 with 2 supplements see all
Effects of Txnip on retinitis pigmentosa (RP) cone mitochondrial size and function.

(A) Representative transmission electron microscopy (TEM) images of RP cones from P20 rd1 cones transduced with wildtype Txnip, Txnip.C247S, or Txnip.S308A (all are AAV8-RedO-Txnip, ≈1 × 109 vg/eye plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye), and control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). (B) Quantification of mitochondrial diameters from control, Txnip, Txnip.C247S, and Txnip.S308A-transduced cones (same as in A). The number in the curly brackets ‘{}’ indicates the sample size, that is, the number of mitochondria from multiple cones of ≥ 1 retina for each condition (five retinas for control, four for Txnip, two for Txnip.C247S, and one for Txnip.S308A). (C) Images of JC-1 dye staining (indicator of electron transport chain [ETC] function) in live cones of P20 rd1 central retina under different conditions (same as in A). Magenta: J-aggregate, indicating high ETC function; green: JC-1 monomer, low ETC function, used for normalization. H2BGFP channel, the tracer of AAV infected area, is not shown. (D) Quantification of normalized cone JC-1 dye staining (fluorescence intensity of J-aggregate:JC-1 monomer) from live cones in P20 rd1 retinas (same as in A) in different conditions (3–4 images per retina). The number in the square brackets ‘[]’ indicates the sample size, that is, the number of images taken from regions of interest of multiple retinas, in each condition. (E) Images of mitoRFP staining (reflecting mitochondrial function) in Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 × 109 vg/eye, plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye and AAV8-SynP136-mitoRFP, 1 × 109 vg/eye) and control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye plus AAV8-SynP136-mitoRFP, 1 × 109 vg/eye) cones from fixed P20 Parp1+/+ rd1 and Parp1-/- rd1 retinas near the optic nerve head. Magenta: mitoRFP; gray: H2BGFP, for mitoRFP normalization. (F) Quantification of normalized mito-RFP:H2BGFP intensity in different conditions (same as in E) of P20 Parp1 rd1 retinas (four images per retina, near optic nerve head). (G) Images of P50 Parp1+/+ rd1 and Parp1-/- rd1 retinas with H2BGFP (gray)-labeled cones transduced with Txnip.C247S (AAV8-RedO-Txnip, ≈1 × 109 vg/eye plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye) and control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). (H) Quantification of H2BGFP-positive cones within the ½ radius of P50 Parp1+/+ rd1 and Parp1-/- rd1 retinas transduced with Txnip.C247S or control (same as in G). (I) Images of P50 Parp1-/- rd1 retinas with H2BGFP (gray)-labeled cones transduced with Ldhb (AAV8-RedO-Ldhb, 1 × 109 vg/eye, plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye) or H2BGFP only (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). (J) Quantification of H2BGFP-positive cones within the ½ radius of P50 Parp1-/- rd1 retinas transduced with Ldhb or H2BGFP only (same as in I). Error bar: standard deviation. NS: not significant, p>0.05, *p<0.05, **p<0.01, ***p<0.001, **** p< or <<0.0001. Txnip.CS: Txnip.C247S; Txnip.SA: Txnip.S308A. RedO: red opsin promoter; AAV: adeno-associated virus; Ldhb: lactate dehydrogenase b.

Figure 5—source data 1

Differentially expressed genes in cones infected by AAV8-RedO-Txnip (1 × 109 vg/eye plus AAV8-SynP136-H2BGFP, 1 × 109 vg/eye) vs. control (AAV8-SynP136-H2BGFP, 1 × 109 vg/eye) in common between two retinitis pigmentosa strains (rd1 and Rho-/-). RedO: red opsin promoter; AAV: adeno-associated virus.

https://cdn.elifesciences.org/articles/66240/elife-66240-fig5-data1-v2.docx
Figure 5—source data 2

Differentially expressed gene(s) in cones infected by AAV8-RedO-Txnip (1 × 109 vg/eye plus AAV8-SynP136-H2BGFP, 1 × 109 vg/eye) vs. control (AAV8-SynP136-H2BGFP, 1 × 109 vg/eye) in common between two wildtype strains (BALB/c and C57BL6/J). RedO: red opsin promoter; AAV: adeno-associated virus.

https://cdn.elifesciences.org/articles/66240/elife-66240-fig5-data2-v2.docx
Figure 5—source data 3

Cone Txnip mRNA raw reads in the RNA-seq data (from 1000 FACS cones per retina).

Data presented as mean ± SEM (n = sample size, i.e., number of retinas per condition).

https://cdn.elifesciences.org/articles/66240/elife-66240-fig5-data3-v2.docx
Figure 5—source data 4

Cone mRNA raw reads from RNA-seq of all 35 retinas used in the study.

Data presented as mean ± SEM (n = 35 for all). No significant difference in these genes with or without Txnip in each strain.

https://cdn.elifesciences.org/articles/66240/elife-66240-fig5-data4-v2.docx

A previous study identified 15 proteins that interact with Txnip.C247S (Forred et al., 2016). Among these interactors was Parp1, which can negatively affect mitochondria through deleterious effects on the mitochondrial genome (Hocsak et al., 2017; Szczesny et al., 2014), as well as effects on inflammation and other cellular pathways (Fehr et al., 2020). Due to the similarities between the effects of Txnip addition and of Parp1 inhibition on mitochondria, Parp1 was tested for a potential role in Txnip-mediated rescue. Parp1 expression was first examined by IHC and found to be enriched in cone inner segments, which are packed with mitochondria (Hoang et al., 2002), as well as in cone nuclei (Figure 5—figure supplement 1G). Interestingly, when a GFP-Txnip fusion protein was expressed in cones, it also was found in these regions (Figure 1—figure supplement 1B). To test for a role of Parp1, Parp1-/- mice were bred to rd1 mice, and their cone mitochondria were examined by TEM and mitoRFP. Parp1-/- rd1 cones possessed larger mitochondria (Figure 5—figure supplement 1H, I) and higher mitoRFP signals than cones from Parp1+/+ rd1 controls (Figure 5E, F). Addition of Txnip.C247S to Parp1-/- rd1 cones did not alter the mitoRFP signals (Figure 5E, F). When Txnip.C247S was added to Parp1-/- rd1 retinas, cone survival was similar to that of Txnip.C247S-transduced Parp1+/+ rd1 retinas, showing that Txnip-mediated survival does not require Parp1 (Figure 5G, H). Parp1-/- rd1 cone survival also was similar to the Parp1+/+ rd1 cones (Figure 5G, H). The rd1 cone degeneration seemed to be faster in these Parp1+/+ and Parp1-/- mice (on 129S background) for unknown reason(s).

The discordance between improved mitochondria and cone survival in these experiments suggested that mitochondrial improvement alone is not sufficient to prolong cone survival. This is consistent with the observations from transduction with Txnip.S308A, as well as Txnip + siLdhb, both of which failed to prolong rd1 cone survival despite improvements in mitochondria (Figure 2A, B, Figure 5A–D, Figure 5—figure supplement 1C–F). To test if improved cone survival requires enhanced lactate catabolism in addition to mitochondrial improvement, we delivered Ldhb to Parp1-/- rd1 cones. Unlike on Parp1+/+ background (Figure 8—figure supplement 1D), a small but significant improvement in cone survival was observed (Figure 5I, J).

Txnip enhances Na+/K+ pump function and cone opsin expression

The results above suggest that Txnip may prolong RP cone survival by enhancing lactate catabolism via Ldhb, which may lead to greater ATP production by the OXPHOS pathway. Cone photoreceptors are known to require high levels of ATP to maintain their membrane potential, relying primarily upon the Na+/K+ ATPase pump (Ingram et al., 2020). To investigate whether Txnip affects the function of the Na+/K+ pump in RP cones, freshly explanted P20 rd1 retinas were stained with RH421, a fluorescent small-molecule probe for Na+/K+ pump function (Fedosova et al., 1995). Addition of Txnip improved Na+/K+ pump function of these cones in lactate medium as reflected by an increase in RH421 fluorescence (Figure 6A, B), consistent with Txnip enabling greater utilization of lactate as fuel. In RP cones, it is also known that protein expression of cone opsin is downregulated, postulated to be due to insufficient energy supply (Punzo et al., 2009). Compared to control, greater anti-opsin staining was observed in Txnip-transduced rd1 cones at P50 (Figure 6C), further supporting the idea that Txnip improves the energy supply to RP cones.

Effect of Txnip on Na+/K+ ATPase pump function and cone opsin expression in retinitis pigmentosa cones.

(A) Images of live ex vivo RH421 stained cones in P20 rd1 retinas transduced with Txnip.C247S (AAV8-RedO-Txnip C247S, ≈1 × 109 vg/eye plus AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye) and control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye) and cultured in lactate-only medium. Magenta: RH421 fluorescence, proportional to Na+/K+ ATPase function; gray: H2BGFP, tracer of infection. (B) Quantification of normalized RH421 fluorescence intensity from Txnip.C247S-transduced cones relative to control in P20 rd1 retinas cultured in lactate-only medium (same as in A, five images per retina). The number in the square brackets ‘[]’ indicates the sample size, that is, the number of images taken from regions of interest of multiple retinas, in each condition. Txnip.CS: Txnip.C247S. (C) Immunohistochemistry with anti-s-opsin plus anti-m-opsin antibodies in the center of P50 rd1 retinas transduced with Txnip.C247S or control. Green: cone-opsins; gray: H2BGFP, tracer of infection. Error bar: standard deviation. **** p< or <<0.0001. RedO: red opsin promoter; AAV: adeno-associated virus.

Dominant-negative HIF1α improves RP cone survival

If improved lactate catabolism and OXPHOS are at least part of the mechanism of Txnip rescue, RP cone survival might be promoted by other molecules serving similar functions. HIF1α can upregulate the transcription of glycolytic genes (Majmundar et al., 2010). Increased glycolytic enzyme levels might push RP cones to further rely on glucose, rather than lactate, to their detriment if glucose is limited. To investigate whether HIF1α might play a role in cone survival, a WT and a dominant-negative HIF1α (dnHIF1α) allele (Jiang et al., 1996) were delivered to rd1 retinas using AAV. A target gene of HIF1α, Vegfa, which might improve blood flow and thus nutrient delivery (but might also increase inflammation), also was tested. The dnHIF1α increased rd1 cone survival, while WT Hif1a and Vegf each decreased cone survival (Figure 7, Figure 7—figure supplement 1D, E).

Figure 7 with 1 supplement see all
Effect of dominant negative HIF1α and Best1-Txnip.C247S.LL351 and 352AA on retinitis pigmentosa cone survival.

(A) Images of P50 rd1 retinas with H2BGFP (gray)-labeled cones transduced with dnHIF1α (AAV8-RO1.7-dnHIF1α, 1 × 109 vg/eye), Hif1a (AAV8-SynPVI-Hif1a, SynPVI is an alternative cone-specific promoter, 1 × 109 vg/eye), Best1-Txnip.C247S.LL351 and 352AA (Txnip.CS.LLAA, driven by a retinal pigmented epithelium-specific promoter; AAV8, 5 × 108 vg/eye; all including AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye), or control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). (B) Quantification of H2BGFP-positive cones within the ½ radius of P50 rd1 retinas transduced with dnHIF1α, Hif1a, Best1-Txnip.C247S.LL351, and 352AA or control (same as in A). B-Tx.CS.LLAA: Best1-Txnip.C247S.LL351 and 352AA. Error bar: standard deviation. *p<0.05, **p<0.01, ***p<0.001.RedO: red opsin promoter; AAV: adeno-associated virus. 

Txnip effects on GLUT1 levels in the RPE and cone survival

To determine if retention of glucose by the RPE might underlie a glucose shortage for cones (Kanow et al., 2017; Wang et al., 2016), we attempted to reprogram RPE metabolism to a more ‘OXPHOS’ and less ‘glycolytic’ status by overexpressing Txnip or dnHIF1α with an RPE-specific promoter, the Best1 promoter (Esumi et al., 2009). The goal was to increase lactate consumption in the RPE, thus freeing up more glucose for delivery to cones. However, no RP cone rescue was observed (Figure 7—figure supplement 1B), possibly due to a clearance of GLUT1 by Txnip from the surface of RPE cells, which would create a glucose shortage for both the RPE and the cones (Swarup et al., 2019Figure 7—figure supplement 1A). To examine the level of GLUT1 in the RPE following introduction of WT Txnip, or Txnip.C247S.LL351 and 352AA, which should prevent efficient removal of GLUT1 (see background in previous section), IHC for GLUT1 was carried out. This assay showed that AAV-Best1-Txnip.LL351 and 352AA did result in less clearance of GLUT1 from the surface of the RPE relative to WT Txnip (Figure 7—figure supplement 1A). Best1-Txnip.C247S.LL351 and 352AA was then tested for rd1 cone rescue, where it was found to improve cone survival (Figure 7), in keeping with the model that the RPE retains glucose to the detriment of cones in RP.

Combination of Txnip.C247S with other rescue genes provides an additive effect

Finally, as our goal is to provide effective, generic gene therapy for RP, and potentially other diseases that affect photoreceptor survival, we used combinations of AAVs that encode genes that we have previously shown prolong RP cone survival and vision. The combination of Txnip.C247S expression in cones, with expression of Nrf2, a gene with anti-oxidative damage and anti-inflammatory activity, in the RPE, provided an additive effect on cone survival relative to either gene alone (Figure 8A, B). This combination also preserved a structure resembling cone outer segments. In WT cones, opsin protein is localized to the outer segment, where photon detection and phototransduction take place. During degeneration in RP, the cone outer segments collapse, and opsin is mislocalized to the plasma membrane (Figure 8C, Figure 8—figure supplement 1A). The combination of RedO-Txnip.C247S and Best1-Nrf2 led to the localization of opsin protein to the outer segment-like structure, rather than to the plasma membrane. An additional morphological phenotype that is especially prominent in the FVB rd1 strain is that of ‘craters’ in the photoreceptor layer. 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 8AWu 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 and 352AA increased, the FVB-specific retinal craters, while both vectors prolonged RP cone survival (Figure 7A). The significance of these craters is thus unclear, as is the mechanism of their formation, though these data point to an origin within the RPE.

Figure 8 with 1 supplement see all
Effect of combinations of Txnip.C247S with Best1-Nrf2 or Tgfb1 on retinitis pigmentosa cone survival.

(A) Images of P50 rd1 retinas with H2BGFP (gray)-labeled cones transduced with Nrf2 (AAV8-Best1-Nrf2, 2.5 × 108 vg/eye), Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 × 109 vg/eye), Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 × 109 vg/eye) + Best1-Nrf2 (AAV8-Best1-Nrf2, 2.5 × 108 vg/eye), or control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). All experimental vector injections included AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye. (B) Quantification of H2BGFP-positive cones within the ½ radius of P50 rd1 retinas transduced with Best1-Nrf2, Txnip.C247S, Txnip.C247S + Best1-Nrf2, or control. Txnip.CS: Txnip.C247S. B-Nrf2: Best1-Nrf2. (C) Immunohistochemistry with anti-S-opsin plus anti-M-opsin antibodies in the center of P130 rd10 retinas transduced with Txnip.C247S (left panel) or Txnip.C247S + Best1-Nrf2 (right panel). Green: cone-opsins; gray: H2BGFP, tracer of infection. (D) Images of P50 rd1 retinas with H2BGFP (gray)-labeled cones transduced with Tgfb1 (AAV8-RedO, 1 × 109 vg/eye), Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 × 109 vg/eye), Txnip.C247S (AAV8-RedO-Txnip.C247S, 1 × 109 vg/eye) + Tgfb1 (AAV8-RedO-Tgfb1, 1 × 109 vg/eye), or control (AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye). All experimental vector injections included AAV8-RedO-H2BGFP, 2.5 × 108 vg/eye. (E) Quantification of H2BGFP-labeled cones within the ½ radius of P50 rd1 retinas transduced with control, Tgfb1, Txnip.C247S, or Txnip.C247S + Tgfb1. Error bar: standard deviation. NS: not significant, p>0.05, *p<0.05, **p<0.01. RedO: red opsin promoter; AAV: adeno-associated virus.

An additional combination that was tested was AAV-RedO-Txnip.C247S with AAV-RedO-Tgfb1, an anti-inflammatory gene that our previous studies showed could eliminate the craters on its own (Wang et al., 2020). This combination did not improve cone survival beyond that of Txnip alone, but almost completely eliminated the craters in an additive fashion with Txnip (Figure 8D, E). In addition, other genes (Hk2, Ldhb, and Cx3cl1) and Nrf2 were expressed specifically in cones in combination with WT Txnip, but did not provide any obvious improvement over Txnip alone (Figure 8—figure supplement 1).

Discussion

Photoreceptors have been characterized as being highly glycolytic, even under aerobic conditions, as originally described by Warburg, 1925. Glucose appears to be supplied primarily from the circulation, via the RPE, which has a high level of GLUT1 (Gospe et al., 2010). Photoreceptors, at least rods, carry out glycolysis to support anabolism, to replace their outer segments (Chinchore et al., 2017), and contribute ATP, to run their ion pumps (Okawa et al., 2008). If glucose becomes limited, as has been proposed to occur in RP, cones may have insufficient fuel for their needs. To explore whether we could develop a therapy to address some of these metabolic shortcomings in RP, we delivered many different types of genes that might alter metabolic programming. From these, Txnip had the strongest improvement on cone survival and vision (Figure 1, Figure 1—figure supplements 1 and 2). This was surprising as Txnip has been shown to inhibit glucose uptake by binding to and aiding in the removal of GLUT1 from plasma membrane (Wu et al., 2013). Moreover, it inhibits the anti-oxidation proteins, the thioredoxins, again by direct binding (Junn et al., 2000; Nishinaka et al., 2001; Nishiyama et al., 1999), so would have been predicted to increase oxidative damage and therefore decrease cone survival. The results with Txnip in its WT form, and from the study of several mutant alleles, provide some insight into how it might benefit cones. The Txnip.C247S allele prevents binding to thioredoxins and gave enhanced cone survival relative to WT Txnip (Figure 2, Figure 2—figure supplement 1). We speculate that, by being free of this interaction, the C247S mutant protein may be more available for other Txnip-mediated activities. In addition, thioredoxin may be made more available for its role in fighting oxidative damage following transduction of the C247S mutant, rather than the WT Txnip allele.

The mechanisms by which Txnip might benefit cones are not fully known, but a study of Txnip’s function in skeletal muscle suggested that it plays a role in fuel selection (DeBalsi et al., 2014). If glucose is limited in RP, then cones may need to switch from a reliance on glucose and glycolysis to an alternative fuel(s), such as ketones, fatty acids, amino acids, or lactate. Cones express oOxct1 mRNA (Shekhar et al., 2016), which encodes a critical enzyme for ketone catabolism, suggesting that cones are capable of ketolysis. In addition, a previous study showed that lipids might be an alternative energy source for cones by β-oxidation (Joyal et al., 2016). It is likely that cones can use these alternative fuels to meet their intense energy demands (Ingram et al., 2020Figure 6). However, the Txnip rescue did not depend on ketolysis or β-oxidation (Figure 3). Due to the diversity of amino acid catabolic pathways, we did not study whether these pathways were required for Txnip’s rescue effect. However, we did discover that Ldhb, which converts lactate to pyruvate, was required. This is an interesting switch as photoreceptors normally have high levels of Ldha (Figure 5—source data 4) and produce lactate (Chinchore et al., 2017; Kanow et al., 2017). An important factor in the reliance on Ldhb could be the availability of lactate, which is highly available from serum (Hui et al., 2017). Lactate could be transported via the RPE and/or Müller glia, and/or the internal retinal vasculature that comes in closer proximity to cones after rod death. Ketones are usually only available during fasting, and lipids are hydrophobic molecules that are slow to be transported across the plasma membranes. Moreover, lipids are required to rebuild the membrane-rich outer segments, and thus might be somewhat limited. Ldhb is not sufficient, however, to delay RP cone degeneration as its overexpression did not promote RP cone survival.

Txnip-transduced RP cones also had larger mitochondria with a greater membrane potential and likely were able to use the pyruvate produced by Ldhb for greater ATP production via OXPHOS. Indeed, Txnip-transduced cones had an enhanced ATP:ADP ratio (Figure 4). However, healthier mitochondria were not sufficient to prolong RP cone survival. Txnip.S308A led to larger mitochondria than control mitochondria as seen by TEM, as well as brighter JC-1 staining and mitoRFP signals, which are indicators of better mitochondrial health, but this allele did not induce greater cone survival (Figure 5, Figure 5—figure supplement 1). Moreover, as Txnip has been shown to interact with Parp1, which can negatively affect mitochondria, we investigated if Parp1 knockout mice might have cones that survive longer in RP. Indeed, the Parp1 knockout mitochondria appeared to be healthier, but Parp1 knockout retinas did not have better RP cone survival than Parp1-WT rd1 retinas (Figure 5, Figure 5—figure supplement 1). In addition, cone rescue by Txnip was not changed in the Parp1 knockout retinas (Figure 5).

The well-described effects of Txnip on the removal of GLUT1 from the cell membrane might seem at odds with the promotion of cone survival. It could be that removal of GLUT1 from the plasma membrane of cones forces the cones to choose an alternative fuel, such as lactate, and perhaps others too. Interestingly, as GLUT1 knockdown was not sufficient for cone survival, Txnip must not only lead to a reduction in membrane-localized GLUT1, but also potentiate a fuel switch, via an unknown mechanism(s) that at least involves an increase of Ldhb activity. A reduction in glycolysis might also lead to a fuel switch. Introduction of dnHIF1α, which should reduce expression of glycolytic enzymes, also benefited cones, while introduction of WT HIF1α did not (Figure 7). HIF1α has many target genes and may alter pathways in addition to that of glycolysis, thus also potentiating a fuel switch once glycolysis is downregulated. An additional finding supporting the notion that the level of glycolysis is important for cone survival was the observation that AAV-Pfkm plus AAV-Hk1 led to a reduction in cone survival (Figure 1—figure supplement 1E). Phosphorylation of glucose by Hk1 followed by phosphorylation of fructose-6-phosphate by the Pfkm complex commits glucose to glycolysis at the cost of ATP. These AAVs may have promoted the flux of glucose through glycolysis, which may have inhibited a fuel switch, and/or depleted the ATP pool, for example, if downstream glycolytic intermediates were used for anabolic needs so that ATP production by glycolysis did not occur, and pyruvate was not produced for conversion to acetyl CoA and ATP production by the mitochondria.

The observations described above suggest that at least two different pathways are required for the promotion of cone survival by Txnip (Figure 9). One pathway requires lactate utilization via Ldhb, but as Ldhb was not sufficient, another pathway is also required. As greater mitochondrial health was observed following Txnip transduction, a second pathway may include the effects on mitochondria. This notion is supported by the observation that the addition of Ldhb to Parp1-/- rd1 cones, which have healthier mitochondria, led to improved cone survival (Figure 5). Txnip alone may be able to promote cone health by impacting both lactate catabolism and mitochondrial health. There may be additional pathways required as well.

Model for the mechanism of Txnip-mediated cone rescue.

The data suggest that at least two pathways are required for Txnip rescue – Pathway I: enhancement of lactate catabolism, which requires the function of LDHB; and pathway II: improved mitochondrial health, possibly through mitochondrial Parp1 inhibition. An additional pathway, pathway III: inhibiting glycolysis by removing glucose transporter 1 (GLUT1) from cell surface, may partially contribute to rescue by working with improved lactate metabolism and improved mitochondrial health, and/or unidentified pathways. This diagram was created from a BioRender Template. MCT: monocarboxylate transporter; 3PG: 3 phosphoglyceric acid; TCA: tricarboxylic acid cycle.

The effects of Txnip alleles expressed only in the RPE provide some support for the hypothesis that the RPE transports glucose to cones for their use, while primarily using lactate for its own needs (Kanow et al., 2017; Swarup et al., 2019). Lactate is normally produced at high levels by photoreceptors in the healthy retina. When rods, which are 97% of the photoreceptors, die, lactate production goes down dramatically. The RPE might then need to retain glucose for its own needs. Introduction of an allele of Txnip, C247S.LL351 and 352AA, to the RPE provided a rescue effect for cones, while introduction of the WT allele of Txnip to the RPE did not. The LL351 and 352AA mutations lead to a loss of efficiency of the removal of GLUT1 from the plasma membrane, while the C247S mutation might create a less glycolytic RPE. The combination of these mutations might then allow more glucose to flow to cones. The untreated RP cones seem to be able to use glucose at a high concentration for ATP production, at least in freshly explanted retinas (Figure 4A). These findings are also consistent with the reported mechanism for cone survival promoted by RdCVF, a factor that is proposed to improve glucose uptake by RP cones, which might be important if glucose is present in low concentration due to retention by the RPE (Aït-Ali et al., 2015; Byrne et al., 2015).

As cones face multiple challenges in the degenerating RP retina, we tested Txnip in combination with genes that we have found to promote cone survival via other mechanisms. The combination of Txnip with vectors fighting oxidative stress and inflammation (AAV-Best1-Nrf2) supported greater cone survival than any of these treatments alone. 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. This finding is in keeping with the interdependence of photoreceptors and the RPE, which is undoubtedly important not only in a healthy retina, but in disease as well.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)Pde6brd1Charles River; TaconicStock #: 207; FVB/NTac.
MGI: 1856373
Genetic reagent (M. musculus)Pde6brd10Jackson LaboratoryStock #: 004297 MGI:2388259
Genetic reagent (M. musculus)Rho-/-Janis Lem (Tufts University)MGI:2680822PMID:9892703
Genetic reagent (M. musculus)Parp1-/-Jackson LaboratoryStock #: 002779. MGI:1857862
AntibodyRabbit anti-GLUT1Alpha DiagnosticsGT11-AIHC (1:300)
AntibodyRabbit anti-PARP1Abcamab227244IHC (1:300)
AntibodyChicken anti-GFPAbcamab13970IHC (1:1000)
AntibodyGoat anti-FLAGAbcamab1257IHC (1:2000)
AntibodyRabbit anti-ARR3Millipore SigmaAB15282IHC (1:1000)
AntibodyRabbit anti-OPN1MWMillipore SigmaAB5405IHC (1:600)
AntibodyRabbit anti-OPN1SWMillipore SigmaAB5407IHC (1:200)
Genetic reagent (M. musculus)Txnip cDNAGeneCopoeiaCat. #: Mm07552 NCBI: NM_001009935.2
Genetic reagent (M. musculus)Hif1a cDNAGeneCopoeiaCat. #: Mm30422 NCBI: NM_010431.2
Genetic reagent (M. musculus)Hk2 cDNAGeneCopoeiaCat. #: Mm03044 NCBI: NM_013820.3
Genetic reagent (M. musculus)Ldha cDNAGeneCopoeiaCat. #: Mm28710 NCBI: NM_001136069.2
Genetic reagent (M. musculus)Ldhb cDNAGeneCopoeiaCat. #: Mm03608 NCBI: NM_008492.2
Genetic reagent (M. musculus)Slc2a1 cDNAGeneCopoeiaCat. #: Mm21137 NCBI: NM_011400.3
Genetic reagent (M. musculus)Bsg1 cDNAGeneCopoeiaCat. #: Mm01471 NCBI: NM_009768.2
Genetic reagent (M. musculus)Cpt1a cDNAGeneCopoeiaCat. #: Mm20470 NCBI: NM_013495.2
Genetic reagent (M. musculus)Oxct1 cDNAGeneCopoeiaCat. #: Mm08941 NCBI: NM_024188.6
Genetic reagent (M. musculus)Mpc1 cDNAGeneCopoeiaCat. #: Mm41054 NCBI: NM_001364919.1
Genetic reagent (M. musculus)Mpc2 cDNAGeneCopoeiaCat. #: Mm19410 NCBI: BC018324.1
Genetic reagent (Homo sapiens)Nrf2 cDNAGeneCopoeiaCat. #: T3128 NCBI: NM_006164.4
Genetic reagent (H. sapiens)Hk1William Hahn and David Root (via Addgene)Cat. #: 23730PMID:21107320
Genetic reagent (H. sapiens)PfkmWilliam Hahn and David Root (via Addgene)Cat. #: 23728PMID:21107320
Genetic reagent (H. sapiens)Pkm2William Hahn and David Root (via Addgene)Cat. #: 23757PMID:21107320
Genetic reagent (H. sapiens)Pkm1Lewis Cantley and Matthew Vander Heiden (via Addgene)Cat. #: 44241PMID:18337815
Recombinant DNA reagentDsRed2-mitoAddgene (Michael Davidson)55838
Recombinant DNA reagentGFP-TxnipAddgene (Clark Distelhorst)18758PMID:16301999
Recombinant DNA reagentPercevalHRAddgene (Gary Yellen)49082PMID:24096541
Recombinant DNA reagentpAAV-RedOBotond RoskaPMID:20576849
Recombinant DNA reagentpAAV-SynPVI (ProA7)Botond RoskaPMID:31285614
Recombinant DNA reagentpAAV-SynP136 (ProA1)Botond RoskaPMID:31285614
Software, algorithmRP cone countingThis paperMATLAB scripts available at https://github.com/sawyerxue/RP-cone-count

Animals rd1 mice were the albino FVB strain, which carries the Pde6brd1 allele (MGI: 1856373). BALB/c, CD1, and FVB mice were purchased from Charles River Laboratories. Due to availability, some of the FVB mice were purchased from Taconic, and we did not notice any difference between the two sources in terms of cone degeneration rate. C57BL/6J, rd10, and Parp1-/- mice were purchased from The Jackson Laboratories and bred in house. C57BL/6J and rd10, which is on C57BL/6J background, carry a null mutation in the Nnt gene (Freeman et al., 2006), but not in the other strains (i.e., rd1, Rho-/-, Parp1-/-, CD1, and BALB/c). We crossed the Parp1-/- mice with FVB mice to generate homozygous Parp1-/- rd1 and Parp1+/+ rd1 mice. Genotyping of these mice was done by Transnetyx (Cordova, TN). The Rho-/- mice were kindly provided by Janice Lem (Tufts University, MA) (Lem et al., 1999).

AAV vector design, authentication, and preparation

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Detailed information of all AAVs used in this study is listed in Figure 1—source data 1, along with the authentication information. cDNAs of mouse Txnip, Hif1a, Hk2, Ldha, Ldhb, Slc2a1, Bsg1, Cpt1a, Oxct1, Mpc1, and Mpc2, and human Nrf2, were purchased from GeneCopoeia (Rockville, MD). Mouse Vegf164 cDNA (Robinson and Stringer, 2001) was synthesized through Integrated DNA Technologies (Coralville, IA). We obtained the following plasmids as gifts from various depositors through Addgene (Watertown, MA): Hk1, Pfkm, and Pkm2 (William Hahn and David Root; #23730, #23728, #23757), Pkm1 (Lewis Cantley and Matthew Vander Heiden; #44241), H2BGFP (Geoff Wahl; #11680), mitoRFP (i.e., DsRed2-mito, Michael Davidson; #55838), GFP-Txnip (Clark Distelhorst; #18758), W3SL (Bong-Kiun Kaang; #61463), 3xFLAG (Thorsten Mascher, #55180), and PercevalHR and pHRed (Gary Yellen; #49082, #31473). The cDNA of mouse RdCVF was a gift from Leah Byrne and John Flannery (UC Berkeley, CA). iGlucoSnFR was provided under a Material Transfer Agreement by Jacob Keller and Loren Looger (Janelia Research Campus, VA). RedO promoter was provided as a gift, and SynPVI (also known as ProA7) and SynP136 (also known as ProA1) promoters were provided under a Material Transfer Agreement, from Botond Roska (IOB, Switzerland). The Best1 promoter was synthesized by a lab member, Wenjun Xiong, using Integrated DNA Technologies based on the literature (Esumi et al., 2009). Mutated Txnip, dominant-negative HIF1α (Jiang et al., 1996), and RO1.7 promoter (Ye et al., 2016) were created from the Hif1a and RedO plasmids correspondingly in house using Gibson assembly. Of note, we found that the RedO promoter is stronger than SynP136 or SynPVI promoters, but less specific. RedO has a low level of expression in some rods. SynP136 and SynPVI drive expression that is exclusive to cones, that is, no rod expression in keeping with the observation of Roska Lab (Jüttner et al., 2019). SynPVI (0.5 kb) is shorter than SynP136 (2 kb), and is thus better for packaging insert genes that have a large size.

All of the new constructs in this study were cloned using Gibson assembly. For example, AAV-RedO-Txnip was cloned by replacing the EGFP sequence of AAV-RedO-EGFP at the NotI/HindIII sites, with the Txnip sequence, which was PCR-amplified from the cDNA vector adding two 20 bp overlapping sequences at the 5′- and 3′-ends. All of the AAV plasmids were amplified using Stbl3 Escherichia coli (Thermo Fisher Scientific). The sequences of all AAV plasmids were verified with directed sequencing and restriction enzyme digestion. The key plasmids were verified with Next-Generation complete plasmid sequencing (MGH CCIB DNA Core), which is able to capture the full sequence of the ITR regions. The genome sequence of critical AAVs (i.e., AAV8-RedO-Txnip.C247S and AAV8-RedO-Txnip.S308A) was verified with PCR and directed sequencing.

All of the vectors were packaged in recombinant AAV8 capsids using 293 T cells and purified with iodixanol gradient as previously described (Grieger et al., 2006; Xiong et al., 2015). The titer of each AAV batch was determined using protein gels, comparing virion protein band intensities with a previously established AAV standard. The concentration of our AAV production usually ranged from 2 × 1012 to 3 × 1013 gc/mL. Multiple batches of key AAV vectors (e.g., four batches of AAV8-RedO-Txnip and three batches of AAV-RedO-siLdhb(#2)) were made and tested in vivo to avoid any potential batch effects.

shRNA

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The shRNA plasmids of Ldhb, Slc2a1, Oxct1, and Cpt1a were purchased from GeneCopoeia, provided as three or four distinct sequences for each gene, driven by the H1 or U6 promoter. The knockdown efficiency of these candidate shRNA sequences was tested by co-transfecting with CAG-TargetGene-IRES-d2GFP vector in 293 T cells as previously described (Matsuda and Cepko, 2007; Wang et al., 2014). The GFP fluorescence intensity served as a fast and direct read out of the knockdown efficiency of these shRNAs. Using this method, we selected the following sense strand sequences to knock down the targeted genes (Figure 2—figure supplement 2, Figure 3—figure supplements 24): siLdhb(#2) 5′-CCATCATCGTGGTTTCCAACC-3′; siLdhb(#1) 5′-GCAGAGAAATGTCAACGTGTT-3′; siLdhb(#3) 5′-GCCGATAAAGATTACTCTGTG-3′; siSlc2a1(#a) 5′-GGTTATTGAGGAGTTCTACAA-3′; siOxct1(#c) 5′-GGAAACAGTTACTGTTCTCCC-3′; siCpt1a(#c) 5′-GCATAAACGCAGAGCATTCCT-3′; and siNC (control sequence that does not target any known gene) 5′-GCTTCGCGCCGTAGTCTTA-3′. We cloned the entire hairpin sequence (including a 6 bp 5′-end lead sequence 5′-gatccg-3′, a 7 bp loop sequence 5′-TCAAGAG-3′ between sense and antisense strands, and a >7 bp 3′-end sequence 5′-ttttttg-3′) and packaged them into AAV8-RedO-shRNA using Gibson assembly as described above. To maximize the knockdown efficacy using a Pol II promoter in AAV (Giering et al., 2008), no extra base pairs were retained between the RedO promoter and the 5′-end sequence of shRNAs. Due to the lack of an adequate Ldhb antibody, we confirmed the in vivo Ldhb knockdown efficiency of all three AAV8-RedO-siLdhb vectors by co-injection with an AAV8-Ldhb-3xFLAG vector into WT mouse eyes with detection using FLAG immunofluorescence as described in the Histology section (Figure 3—figure supplement 1A).

Subretinal injection

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On the day of birth (P0), AAVs were injected into the eyes of pups as previously described (Matsuda and Cepko, 2007; Xiong et al., 2015). For all experiments in which cones were quantified, and to provide a means to trace infection (e.g., for IHC), 2.5 × 108 vg/eye of AAV8-RedO-H2BGFP was co-injected with the experimental AAVs, or alone as a control. The dose of each experimental AAV was ≈1 × 109 vg/eye, individually or when in combination with other experimental AAV’s, and the injection volume was the same for each injection. For all other experiments, such as FACS sorting and ex vivo live imaging, 1 × 109 vg/eye of AAV8-SynP136-H2BGFP, which provides better cone specificity but lower expression level than RedO-H2BGFP, was co-injected. All of the control groups in this study refer to AAV reporter (e.g., H2BGFP or PercevalHR) injection alone.

Photopic visual acuity measured for optomotor response

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The photopic optomotor response of mice was measured using the OptoMotry System (CerebralMechanics) at a background light of ≈70 cd/m2 as previously described (Xiong et al., 2019). The contrast of the grates was set to be 100%, and temporal frequency was 1.5 Hz. The threshold of mouse visual acuity (i.e., maximal spatial frequency) was tested by an examiner without knowledge of the control vs. experimental groups. During each test, the direction of movement of the grates (i.e., clockwise or counterclockwise) was randomized, and the spatial frequency of each testing episode was determined by the software. Without knowing the spatial frequency of the moving grates, the examiner reported either ‘yes’ or ‘no’ to the system until the threshold of acuity was determined by the software. Optomotor tests were conducted on rd10 and Rho-/- mice, but not with rd1 strain, which loses vision at a very early age, before any meaningful test could be performed.

Electroretinography

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To probe rd10 cone function, photopic ERGs were measured in anesthetized mouse eyes in vivo as previously described using an Espion E3 System (Diagonsys LLC) (Xiong et al., 2019; Xue et al., 2020). Multiple white flashes were applied to elicit ERG responses at 1 (peak), 10 (peak), 100 (xenon), and 1000 (xenon) cd s/m2 intensities with a white light background of 30 cd/m2. A ketamine/xylazine cocktail (100/10 mg/kg) was introduced via intraperitoneal injection to mice for anesthesia. Tropicamide 1% eye solution (Bausch + Lomb) was used to dilate the pupil.

Histology

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Mice were euthanized with CO2 and cervical dislocation, and the eyes were enucleated. For flat-mounts, retinas were separated from the rest of the eye using a dissecting microscope and were fixed in 4% paraformaldehyde solution for 30 min. The retinas were then flat-mounted on a glass slide and coverslip. For H2BGFP-labeled cone imaging, we used a Keyence fluorescence microscope with a ×10 objective (Plan Apo Lamda 10x/0.45 Air DIC N1) and GFP filter box (OP66836).

For cone opsin antibody staining in whole-mount retinas, after fixation, retinas were blocked for 1 hr in PBS with 5% normal donkey serum and 0.3% Triton X-100 at room temperature. After blocking, retinas were incubated with a mixture of 1:200 anti-s-opsin (OPN1MW) antibody (AB5407, EMD Millipore) and 1:600 anti-m-opsin (OPN1SW) antibody (AB5405, EMD Millipore) in the same blocking solution overnight at 4°C, followed by secondary donkey-anti-rabbit antibody staining (1:1000, Alexa Fluor 594) at room temperature for 2 hr, then flat-mounted on a glass slide and coverslip.

For frozen sections, whole eyes were fixed in 4% paraformaldehyde solution for 2 hr at room temperature, followed by removal of the cornea, lens, and iris. The eye cups then went through a 15% and 30% sucrose solution to dehydrate at room temperature, followed by overnight incubation in 1:1 30% sucrose and Tissue-Tek O.C.T. solution at 4°C. Eye cups were embedded in a plastic mold, frozen in a −80°C freezer, and cut into 20 or 12 μm thin radial cross sections that were placed on glass slides. Antibody staining was done similarly to whole-mounts as described above and previously (Wang et al., 2014). PBS with 0.1% Triton X-100, 5% normal donkey serum ,and 1% bovine serum albumin (BSA) was used as the blocking solution, except for FLAG detection (10% donkey serum and 3% BSA). GLUT1 (encoded by Slc2a1 gene) antibody (GT11-A, Alpha Diagnostics) was used at 1:300 dilution, PARP1 antibody (ab227244, Abcam) was used at 1:300 dilution, GFP antibody (ab13970, Abcam) was used at 1:1000 dilution to detect GFP-Txnip, ARR3 antibody (AB15282, Millipore Sigma) was used at 1:1000 dilution to detect cone arrestin (encoded ay the Arr3 gene), and FLAG antibody (ab1257, Abcam) was used at 1:2000 based on a previous study (Ferrando et al., 2015). If applicable, 1:1000 PNA (CY5 or rhodamine labeled) for cone extracellular matrix labeling and 1:1000 DAPI were used to co-stain with secondary antibodies. Stained sections were imaged with a confocal microscope (LSM710, Zeiss) using ×20 or ×63 objectives (Plan Apo 20x/0.8 Air DIC II, or Plan Apo 63X/1.4 Oil DIC III).

Automated cone counting

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The cone-H2BGFP images of entire flat-mounted retinas were first analyzed in ImageJ to acquire the diameter and the center parameters of the sample. 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 six retinas. The threshold of intensity and variance thus determined were then set at fixed values for all the experiments that used cone quantification. The background intensity did not interfere with the accurate counting on the raw images by this MATLAB script despite the observation that the intensity of some images looked different at low magnification. This method of cone counting was further verified using FACS analysis of cones in this study (Figure 1—figure supplement 2B) and an independent study (Wang et al., 2020).

Live imaging of cones on ex vivo retinal explants

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For JC-1 mitochondrial dye staining, the retina was quickly dissected in a solution of 50% Ham's F-12 Nutrient Mix (11765054, Thermo Fisher Scientific) and 50% Dulbecco's Modified Eagle Medium (DMEM; 11995065, Thermo Fisher Scientific) at room temperature. They were then incubated in a culture medium containing 50% Fluorobrite DMEM (A1896701, Thermo Fisher Scientific), 25% heat-inactivated horse serum (26050088, Thermo Fisher Scientific), and 25% Hanks' Balanced Salt Solution (14065056, Thermo Fisher Scientific) with 2 μM JC-1 dye (M34152, Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator for 20 min. The retinas were washed in 37°C in this medium without JC-1 three times, transferred to a glass-bottom culture dish (MatTek P50G-1.5-30F) with culture medium, and imaged using a confocal microscope (LSM710 Zeiss), which was equipped with a chamber pre-heated to 37°C with pre-filled 5% CO2. Right before imaging, a cover slip (VWR 89015-725) was gently applied to flatten the retina. Regions of interest (with H2BGFP as an indicator of successful AAV infection and to set the correct focal plane on the cone layer) were selected under the eyepiece with a ×63 objective (Plan Apo 63X/1.4 Oil DIC III). Fluorescent images from the same region of interest were obtained with the excitation wavelength of 561 nm (for J-aggregates), 514 nm (for JC-1 monomer), and 488 nm (for H2BGFP). Four different regions of interest from the central part of the same retina were imaged before moving to the next retina.

For RH421 (Na+/K+ ATPase dye) staining, similar steps were taken as for JC-1 staining, with the following modifications: (1) 0.83 μM RH421 dye (61017, Biotium) was added to the glass-bottom culture dishes just before imaging, but not during incubation in the incubator, due to the fast action of RH421. (2) Five regions of interest were imaged per retina from the central area. (3) The dissection and culture medium were lactate-only medium (see below). (4) Excitation wavelengths: 561 nm (RH421) and 488 nm (H2BGFP).

For imaging genetically encoded metabolic sensors (PercevalHR, iGlucoSnFR, and pHRed), retinas were placed in the incubator for 12 min and then taken to confocal imaging without any staining. For the high-glucose condition, the culture medium described above contains ≈15 mM glucose without lactate or pyruvate. For the lactate-only condition, the culture and dissection media were both glucose-pyruvate-free DMEM (A144300, Thermo Fisher Scientific) and were supplemented with 20 mM sodium L-lactate (71718, Sigma-Aldrich). For the pyruvate-only condition, the culture and dissection media were both glucose-pyruvate-free DMEM plus 10 or 20 mM sodium pyruvate (P2256, Sigma-Aldrich). No AAV-H2BGFP was co-injected with these sensors since the sensors themselves could be used to trace the area of infection. The excitation wavelengths for sensors were 488 and 405 nm (PercevalHR, ratiometric high and low ATP:ADP), 488 and 561 nm (iGlucoSnFR, glucose-sensing GFP and normalization mRuby), and 561 and 458 nm (pHRed, ratiometric low and high pH).

The fluorescent intensity of all acquired images was measured by ImageJ. The ratio of sensors/dye was normalized to averaged control results taken at the same condition.

Flow cytometry and cell sorting

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All flow cytometry and cell sorting were performed on MoFlo Astrios EQ equipment. Retinas were freshly dissected and dissociated using cysteine-activated papain followed by gentle pipetting (Shekhar et al., 2016). Before sorting, all samples were passed through a 35 μm filter with buffer containing Fluorobrite DMEM (A1896701, Thermo Fisher Scientific) and 0.4% BSA. Cones labeled with AAV8-SynP136-H2BGFP (highly cone-specific) were sorted into the appropriate buffer for either ddPCR or RNA sequencing.

RNA sequencing

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RNA sequencing was done as previously described (Wang et al., 2019). 1000 H2BGFP-positive cones per retina were sorted into 10 μL of Buffer TCL (QIAGEN) containing 1% β-mercaptoethanol and immediately frozen in −80°C. On the day of sample submission, the frozen cone lysates were thawed on ice and loaded into a 96-well plate for cDNA library synthesis and sequencing. A modified Smart-Seq2 protocol was performed on samples by the Broad Institute Genomics Platform with ∼6 million reads per sample (Picelli et al., 2013). The reads were mapped to the GRCm38.p6 reference genome after quality control measures. Reads assigned to each gene were quantified using featureCounts (Liao et al., 2014). Count data were analyzed using DESeq2 to identify differentially expressed genes, with an adjusted p value less than 0.05 considered significant (Anders and Huber, 2010). The raw results have been deposited to Gene Expression Omnibus (accession number GSE161622 for RP cones and GSE168503 for WT cones).

ddPCR

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RNA was isolated from 20,000 sorted cones per retina using the RNeasy Micro Kit (QIAGEN) as previously described (Wang et al., 2020) and converted to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). cDNA from each sample was packaged in droplets for Droplet Digital PCR (ddPCR) using QX200 EvaGreen Supermix (#1864034). The reads of expression were normalized to the housekeeping gene Hprt. Sequences for RT-PCR primers were designed using the IDT online RealTime qPCR primer design tool. The following primers were selected for the genes of interest: Txnip (forward 5′-ACATTATCTCAGGGACTTGCG-3′; reverse 5′-AAGGATGACTTTCTTGGAGCC-3′), Hprt (forward 5′-TCAGTCAACGGGGGACATAAA-3′; reverse 5′-GGGGCTGTACTGCTTAACCAG-3′), mt-Nd4 (forward 5′-AGCTCAATCTGCTTACGCCA-3′; reverse 5′-TGTGAGGCCATGTGCGATTA-3′), mt-Cytb (forward 5′-ATTCTACGCTCAATCCCCAAT-3′; reverse 5′-TATGAGATGGAGGCTAGTTGGC-3′), mt-Co1 (forward 5′-TCTGTTCTGATTCTTTGGGCACC-3′; reverse 5′-CTACTGTGAATATGTGGTGGGCT-3′), Acsl3 (forward 5′- AACCACGTATCTTCAACACCATC-3′; reverse 5′- AGTCCGGTTTGGAACTGACAG-3′), and Ftl1 (forward 5′- CCATCTGACCAACCTCCGC-3′; reverse 5′- CGCTCAAAGAGATACTCGCC-3′).

Transmission electron microscopy

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Intracardial perfusion (4% PFA + 1% glutaraldehyde) was performed on ketamine/xylazine (100/10 mg/kg) anesthetized mice before the removal of eyes. The cornea was sliced open and the eye was fixed with a fixative buffer (1.25% formaldehyde + 2.5% glutaraldehyde + 0.03% picric acid in 0.1 M sodium cacodylate buffer, pH 7.4) overnight at 4°C. The cornea, lens, and retina were removed before resin embedding, ultrathin sectioning, and negative staining at Harvard Medical School Electron Microscopy Core. The detailed methods can be found on the core’s website (https://electron-microscopy.hms.harvard.edu/methods). The stained thin sections were imaged on a conventional TEM (JEOL 1200EX) with an AMT 2k CCD camera.

Statistics

For the comparison of two sample groups, two-tailed unpaired Student’s t-test was used to test for the significance of difference, except for P140 Rho-/- optomotor assay (paired two-tail t-test). For comparison of more than two sample groups, ANOVA and Dunnett's multiple comparison test were performed in Prism 8 software to determine the significance. A p-value of <0.05 was considered statistically significant. All error bars are presented as mean ± standard deviation, except for the rd10 optomotor assays, ERG, FACS cell %, and RNA-seq raw reads (mean ± SEM).

Study approval

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All animal experiments were approved by the IACUC of Harvard University in accordance with institutional guidelines.

Data availability

Sequencing data have been deposited in GEO under accession codes GSE161622 and GSE168503.

The following data sets were generated
    1. Xue Y
    2. Cepko CL
    (2020) NCBI Gene Expression Omnibus
    ID GSE161622. RP mouse cone transcriptom change with Txnip treatment.
    1. Xue Y
    2. Cepko CL
    (2021) NCBI Gene Expression Omnibus
    ID GSE168503. Wildtype (wt) mouse cone transcriptome change with Txnip treatment.

References

    1. Robinson CJ
    2. Stringer SE
    (2001)
    The splice variants of vascular endothelial growth factor (VEGF) and their receptors - PubMed
    Journal of Cell Science 114:853–865.
    1. Wu DM
    2. Ji X
    3. Ivanchenko MV
    4. Chung M
    5. Piper M
    6. Rana P
    7. Wang SK
    8. Xue Y
    9. West E
    10. Zhao SR
    11. Xu H
    12. Cicconet M
    13. Xiong W
    14. Cepko CL
    (2021)
    Nrf2 overexpression rescues the RPE in mouse models of retinitis pigmentosa
    JCI Insight 6:e145029.

Decision letter

  1. Claude Desplan
    Reviewing Editor; New York University, United States
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States
  3. James B Hurley
    Reviewer; University of Washington, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

Your screen of several candidate genes that might make cones more robust to stress caused by genetic deficiencies led to the identification of Thioredoxin-interacting protein (Txnip) that is very effective at prolonging cone survival in models of retinal degeneration. Txnip appears to be switching the fuel source and enhancing lactate catabolism in cone photoreceptors, thus allowing cones to use alternative fuels more effectively. This study has the potential to become a therapeutic approach to treat retinal degeneration

Decision letter after peer review:

Thank you for submitting your article "AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: James B Hurley (Reviewer #1).

The reviewers, who have different expertise, have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The three reviewers agree that the study is significant and might lead to potential clinical applications. However, they also noted some weaknesses that must be addressed. These include missing controls and quantifications that would reinforce the data.

I am sure that you will do your best to address many of the comments from these very thorough reviews but there is a small number of points that must absolutely be addressed:

– You should demonstrate convincingly that the proteins, especially Txnip and GLUT1 are expressed and that the levels of their over-expression compared to endogenous expression are substantial.

– Although you do show a rescue of cone survival, you must address whether the structure and function of these cones are indeed maintained. You should present data about the shape of the outer segments and about the cone synapses (in P50 retina?). Histology, IHC or TEM if feasible.

– Is TXNIP's rescue mechanistically mediated through anti-oxidative stress or mitochondrial biogenesis? Checking the difference in the number of mitochondria in Txnip-treated retina would be helpful.

Reviewer #1: (Recommendations for the authors)

1. Injections of the Txnip gene from AAV with cone-specific promoters slows retinal degeneration. A control that is missing throughout this very interesting and significant report is direct evidence that Txnip actually is being overexpressed. My confidence in the conclusions would be stronger if the authors could show by immunoblot or by IHC that there is normally some endogenous expression of Txnip in cones and that when the eyes have in them AAV carrying a cone-specific promoter and the coding sequence for Txnip that Txnip expression in cones increases substantially. It would be very helpful if the level of over-expression could be quantified.

2. In theory when Txnip is over-expressed in cones there should be a decrease in GLUT1 expression in cones, but not in RPE cells. This report would be stronger if the authors could show by IHC that cone expression of GLIT1 is diminished in the Txnip cone OE retinas but GLUT1 would not be diminshed in the cones that express the mutant Txnip that is incapable of stimluating endocytosis of GLUT1.

3. Lines 259-261. The finding that expression of ldhb prolongs survival of cones in these and other experiments throughout the report is interesting. It implies that cones are using lactate. But I would expect when rods are gone there is not much lactate in the retina. Please check that – i.e. measure lactate levels in the WT and rd1 retinas. The assay could be done with a commercial kit. If the lactate levels are low in the rd1 retina then it is puzzling why enhanced ldhb expression prolongs survival. Please address this either here or in the discussion. Also, it would be helpful to confirm with an antibody that ldhb is being expressed (in the treated retinas at higher than normal levels).

4. I think readers would like to know what happens to the phosphorylation state of mTOR in cones in these experiments and I think it would be relatively simple to test. In some cells phosphorylation of mTOR in the absence of glucose can be harmful, I think because it is prepping cells for a metabolic state that does not exist in the tissue. The Cepko lab reported previously that mTOR phosphorylation in cones is diminished in rd mutants, consistent with a compromised supply of glucose to the retina. What effect does Txnip expression in cones have on mTOR phosphorylation (diminish, increase or no effect) and what effect does combined over-expression of HK1 and PFK1 expression have on mTOR phosphorylation?

5. Lines 277-286. Please confirm by IHC or immunoblot that the wt or dnHIF1 is over-expressed and, if possible, quantify by how much it is over-expressed.

6. Extended data 6a and lines 300-302. I would have expected GLUT1 to be on the basal and apical surfaces of the Txnip.CS.LLAA mutant overexpressor but it seems to be only on the apical surface. Please mention and discuss this.

Reviewer #2 (Recommendations for the authors):

The manuscript includes extensive data. However, several points require clarification and the text and figures need consistency. Specific comments/suggestions are as follows:

The authors concluded previously that cone starvation and degeneration are due to shortage of glucose in four animal models of RP (Punzo et al., 2009). However, except rho-/-, which is not the focus in this manuscript, the rest three animal models were not used in this study. It is unclear whether rd1 and rd10 also have shortage of ATP and how severe the shortage is as compared to the WT, or it is simply more ATP can better maintain cone survival.

The authors claim the treatment "correlates with the presence of a healthier mitochondria". As shown in Figure 5a, although the size of mitochondria was bigger in the treated groups compared to the control, those mitochondria did not seem to harbor a typical morphology. More transmission electron microscopy (TEM) images of different magnitudes are needed to show the localization of these mitochondria as well as the morphology of more mitochondria. Without the images from WT, it is hard to evaluate whether it is due to strain of the mice or other factors, or how a healthy mitochondrion should look like. Likewise, a much higher membrane potential in mitochondria does not necessarily mean healthier mitochondria. Additionally, the authors should show the mitochondria morphology of P50 rd1 as well, as that's the end point of the whole experiments and can provide useful information on the duration of the treatment.

Although the flat mount images in this manuscript show the overall cell survival in the whole retina, immunostaining of retinal sections is needed to address important questions related to retinal functions such as the structure of the retina, the morphology of the survival cone photoreceptors and the subtypes of the surviving cones, i.e., whether Txnip can maintain both S- and M- cones. The authors mentioned "this combination also preserved the RP cone outer segments" (line 312); yet, it is hard to evaluate the outer segment by flat mount, although a slight difference may be noticeable between the two groups.

The automated method for cone quantification (1/2 radius cell count) that counts H2BGFP-positive cones in the central half of the retina (previous calculation of the diameter of flat mount retinas) raises some concerns. Is the area of the retina in the 1/2 radius the same between samples? Not all flat-mounted retinas look the same (differences due to retina dissection), and this can influence the area used to count cones. In addition, is this method counting all the cones present in the sample? Are all cones H2BGFP-positive? What is the viral transduction efficiency (alone and co-injecting two AAVs)? Is it the same between samples? Are authors using z-stack confocal images for quantification?

It is appropriate to quantify density of cones (number of cones/mm2) in defined areas of interest, which should be the same for all the samples. To achieve this, a proper orientation of the whole-mounted retina is necessary, and this information is lacking in the images included in the manuscript.

The authors could consider the quantification of "holes" or "craters" in the cone mosaic as an indicator of differential treatment effect.

The authors do not quantify the level of Txnip expression in transduced retinas. It would be important to know the level of overexpression they have achieved and compare it with the expression in untreated RP retinas. In addition, it would be relevant to know if treatment effects correlate with Txnip expression levels.

OMR (based on head movements of mice observed by a human observer) can't be directly equated to visual acuity (see PLoS ONE 8(11): e78058, 2013; J Neurophysiol 118: 300-316, 2017). One needs to interpret such data with caution. Is it possible to do multi-focal ERG?

The authors confirmed the effect of Txnip on cone survival using three different animal models. However, in some follow-up experiments, the authors used these models interchangeably and it leads to confusion. For example, while rd1 is the main focus of the manuscript, the authors did not show the rescued visual acuity of these mice. Even if there is no effect of Txnip on visual acuity in these mice, the authors should still mention this to provide some information of the effectiveness of the treatment on some fast-degenerating mouse models. In addition, the authors used rd10 and rho-/- as well in the manuscript. Did the authors test the mechanisms they found using rd1 on rd10 and rho-/-? These data would be helpful to discriminate whether the switch of energy source by Txnip in the survival of cone cells is specific to rd1.

Figure 8a and 8b described the effect of vector combinations on cone survival in rd1, whereas Figure 8c actually mentioned the effect on outer segment in rd10. The authors did not even mention this switch of animal models in the text. The effect on outer segment in rd10 mice does not mean it would surely give the same effect in rd1 mice. It is unclear whether there was no improvement of outer segment in treated rd1 mice, or the vector combination did not have any effect on cone survival in rd10 mice.

Reviewer #3 (Recommendations for the authors):

In order to strengthen this manuscript, the following suggestions should be addressed:

1. Did the authors observe the difference in mice with or without NNT deletion? Is there an NNT deletion in rd1, rd10, parp1-/-, and rho-/- mice?

2. Extended Data Figure 1b and 1d: to show AAV8-RO1.7-GFP expression in cone photoreceptors, the Arr3 antibody will be better than PNA since PNA could only express in the cone outer and inner segments. Extended Data Figure 1b showed some GFP in the cytoplasm, is this the H2B-EGFP?

3. Figure 1b: are these numbers within the 1/2 radius or within selected small boxes? If these numbers are from selected small boxes, how many small boxes from each were selected? The authors should explain how they selected the small boxes as there is some variation on the dorsal retina or ventral retina. For example, in P130 rd10, the cone cell densities are quite different in the four leaves of the inner circle. Same question for the P50 rd1, there is some area with a patchy absence of cone cells. If the numbers are within 1/2 radius, why use 1/2 radius rather than 2/3 or 1/3 radius?

4. It would be helpful if the authors explained the reason behind each time point, i.e., P50 for rd1, P130 for rd10, and P150 for Rho -/-.

5. Figure 5i: the number of cone cells at 1/2 radius in the whole mount retina looks quite similar, but it seems the number of cone cells in the periphery of control retina is more than the number in the +Ldhb retina.

6. Bracket "[]" in Figure 4, and curly bracket "{}" in Figure 5 indicate sample size. Are these the sample sizes of the image/retina or samples sizes of the mice?

7. Figure 5: it is unclear how reliable the Mt size is, because the EM images are not SBF-SEM. The shape and morphology of mitochondria is in 3D and the EM images are taken from a different orientation and thus may have different results.

8. Did the authors observe any difference in the number of mitochondria in Txnip-treated retina?

9. Does Txnip increase mitochondrial biogenesis?

10. It would be better if the authors could clarify the meaning of n.d. in "Wu et al., n.d.".

11. Line 336-337: it remains unclear what the role of anti-oxidative stress is in the Txnip-mediated cone rescue effect. It would be helpful if the authors added an experiment to demonstrate the changes in ROS in Txnip (wt and C237S)-mediated activities.

12. Line 353: it would be helpful if the authors could explain why the Txnip rescue did not depend on ketolysis or ß-oxidation.

13. Line 487-488: "The GFP fluorescence intensity served as a fast 488 and direct read out of the knockdown efficiency of these shRNAs." The GFP after IRES can sometimes too weak to produce a fast and direct readout. Do the authors have the image/a figure on this?

14. It is unclear whether the control group in this study refers to the non-injected eye or to saline injections.

https://doi.org/10.7554/eLife.66240.sa1

Author response

The three reviewers agree that the study is significant and might lead to potential clinical applications. However, they also noted some weaknesses that must be addressed. These include missing controls and quantifications that would reinforce the data.

I am sure that you will do your best to address many of the comments from these very thorough reviews but there is a small number of points that must absolutely be addressed:

– You should demonstrate convincingly that the proteins, especially Txnip and GLUT1 are expressed and that the levels of their over-expression compared to endogenous expression are substantial.

Since our gene therapy is highly specific to a small population of cells in a heterogenous tissue (i.e. cones in the degenerating retina), the only protein detection method that might work would be IHC. We had repeatedly tried IHC for Txnip with two commercially available antibodies at the early stage of this study. The best images are shown in Author response image 1, but are not convincing. The best of the available Txnip antibodies (Thermo Fisher #40-3700) was able to detect Best1-Txnip overexpression in the RPE, where there is low staining in the uninfected tissue and high vector expression from the Best1 promoter (Author response image 1A,B). However, this antibody gave ambiguous staining in the retina (Author response image 1C-F). There is almost no Txnip RNA in cones (new Figure 5—source data 3), almost none in rods (Drop-seq data from Shekhar et al., 2017), and a very low signal in a small fraction of Mueller glia (Dropseq data from Shekhar et al., 2017). Yet, quite a bit of IHC signal was detected in the outer nuclear layer and inner and outer segment layers in the uninfected retina, with no obvious greater IHC signal in the AAV-Txnip infected retina. Due to these problems, we made a fusion protein of GFP-Txnip and used AAV8-RedO1.7 to express it, in both RP and WT retinas (Figure 1—figure supplement 1B, originally it was shown in Extended Data Figure 1b). We found that GFP-Txnip was excluded from cone outer segments, but was located in all other regions, including in the reported locations of a protein with which it is reported to interact (PARP1), in the inner segments and nucleus.

Author response image 1

For GLUT1 IHC, the antibody was not the issue. We had shown significant removal of GLUT1 from the RPE by Best1-Txnip in the original submission (Figure 7—figure supplement 1, originally Extended Data Figure 6a; Author response image 2A,B). However, since GLUT1 is a membrane protein and cones are surrounded by the processes of neighboring cells (i.e. rods, Muller glia, ad RPE in WT retina; Muller glia and RPE processes in the RP retina after rods are gone), and because all of these other cell types express quite a bit of GLUT1, we had trouble distinguishing GLUT1 IHC signals within cones (Author response image 2C,D at the end of this letter). For this reason, we could not tell if there was any drop in cone GLUT1 IHC by RedO-Txnip (Author response image 2E,F). Moreover, the raw reads from our RNA-seq dataset suggested that the GLUT1 (i.e. Slc2a1) level was relatively low in cones (new Figure 5—source data 4). Therefore, we did not think that we would be able to observe a significant GLUT1 reduction in Txnip-treated cones, as was observed in the RPE.

Author response image 2

We did not include these IHC results given that they were inconclusive. In addition to the RNA reads for GLUT1, we have included a table of Txnip raw reads from our cone RNAseq data (GEO depositions: GSE161622 and GSE168503) of Txnip-treated vs. control of four different strains (new Figure 5—source data 3). These numbers clearly demonstrate >100 fold increase of Txnip mRNA through addition of RedO-Txnip in all strains. The Txnip ddPCR data confirmed this finding (Figure 5—figure supplement 1B, originally Extended Data Figure 5b). As mentioned above, there is almost no Txnip mRNA in wildtype or RP control cones, so the fold amplification is huge. Although these data are for RNA, and not protein, (and we would have loved to be able to show protein), the only alternative hypothesis to protein expression leading to rescue is that the AAV DNA, encoding Txnip, or the mRNA for Txnip, caused the rescue. These are highly unlikely scenarios given that we made several alleles of Txnip that had only base pair changes (e.g. C247S, S308A). These base pair changes affected the rescue activity. The most likely hypothesis is that these base pair changes resulted in protein changes, as otherwise, we would have to conclude that base pair changes to the AAV DNA or mRNA could both increase (C247S) and decrease rescue (S308A). These base pair changes were chosen as the literature had shown that the corresponding amino acid changes changed the activity and binding partners for the Txnip protein.In addition to the efforts described above, we validated the DNA sequences of our AAV vectors. (Figure 1—source data 1, originally Supplementary Table 1, and Methods) using several methods.

– Although you do show a rescue of cone survival, you must address whether the structure and function of these cones are indeed maintained. You should present data about the shape of the outer segments and about the cone synapses (in P50 retina?). Histology, IHC or TEM if feasible

We did not observe any obvious structural changes to either RP or WT cones by Txnip, and we have not claimed any RP cone structural benefits in any part of our text. In fact, we have never seen nice outer segments or wildtype cone-like morphology for any of our rescue treatments, nor from those of other laboratories. We have seen more opsin protein, and a bit of an outer segment structure with Txnip C247S (Figure 6C). Our best guess is that, given the complete collapse of the outer nuclear layer (ONL), the cones are challenged by mechanical/physical changes in the architecture of the ONL/RPE interface. In addition, there are data from our lab and others that there are many problems for cones in the RP environment, including oxidative stress, inflammation, and metabolic challenges. Our lab has now assayed >50 genes to address these challenges, and we have been publishing some of the successes (Punzo et al. 2009, Xiong et al. 2015, Wang et al. 2019, 2020, Chinchore et al. 2019, Wu et al. 2021). Our goal is to combine treatments to try to combat multiple problems for cones. Consistent with this hypothesis, we did achieve greater rescue by combining RedOTxnip (for cone metabolism) with Best1-Nrf2 (to combat oxidative damage and possibly inflammation in the RPE) (Figure 8A,B). We believe that the most important way to assess a benefit to cones is to show functional benefits, which is the point of the optomotor assay (Figure 1C), where we did see significant benefit due to Txnip.

However, to address the question of cone morphology, we have added a set of cone arrestin (ARR3) IHC and TEM images to show what we observed (Figure 1—figure supplement 2A, Figure 5—figure supplement 2), as well as IHC for s- and m- opsin separately (Figure 8—figure supplement 1A), and updated the text to address this point. For cone function, we have added our ERG data as Figure 1—figure supplement 2E. The interpretation of this neutral result is in the corresponding point to Reviewer #2 in this letter below.

– Is TXNIP's rescue mechanistically mediated through anti-oxidative stress or mitochondrial biogenesis? Checking the difference in the number of mitochondria in Txnip-treated retina would be helpful.

Mitochondria are thought to be one continuous structure, although when seen in cross section they appear to be independent units. It is thus hard to quantify their numbers. The staining with mitoRFP is the best proxy we could come up with to quantify the Txnip effect on “numbers” of mitochondria, as we reported in the initial submission (Figure 5—figure supplement 1D, originally Extended Data Figure 5d). It is also obvious from cross sections that the Txnip-treated cells have larger mitochondrial cross sections, as quantified in Figure 5B. The TEM images that we showed in the first submission, along with the quantification of the average size of the cross sections, show that Txnip increases the size. We added 22 more TEM images to share our observations on the mitochondrial morphology in the cones from both WT and RP retinas (new Figure 5—figure supplement 2B-D). We found it difficult to compare mitochondria in wildtype healthy inner segments/ OPL to those in degenerating cones due to the collapse of the normal cone morphology i.e. there are no organized inner segments in RP cones, which is where most mitochondria localize in wild type cones.

We observed that Txnip did not upregulate the mitochondrial ETC genes in WT cones as it did in RP cones. Txnip is thus unlikely to have as its constitutive role mitochondria biogenesis. We have added this information to the revised manuscript (new Figure 5—figure supplement 2A, and Figure 5—source data 2). This additional WT cone RNA-seq results have been recently deposited to GEO (accession number GSE168503). However, it is hard to know whether the benefits to RP cone mitochondria are due to increased biogenesis or other effects. Cone degeneration in the rd10 strain has been reported to be accompanied by swollen and abnormal mitochondria, which are rescued by a RipK3 antagonist (Murakami et al., 2012, PNAS). As this drug can inhibit necrotic death due to oxidative stress, which is mediated by RipK3, we understand that this drug, as well as Txnip, might protect cones via inhibition of oxidative damage or death induced by oxidative stress. Of interest regarding this possibility is that the Txnip C247S allele does not bind thioredoxin, and it rescues better. It could be that by liberating thioredoxin, it allows thioredoxin to increase its anti-oxidation role. Alternatively, or additionally, it could be that Txnip is liberated from thioredoxin to better perform its other role(s) for cone rescue.

Regarding the role of Txnip in oxidative damage, it has been reported to inhibit the thioredoxins when it binds to them, making oxidative damage worse. This would predict that reduction or removal of Txnip would reduce oxidative damage, i.e. an effect in the opposite direction to the benefits that we observe. In keeping with this, Txnip KO Muller glial cells in culture have less mitophagy, in a condition used to model diabetes by culturing in a high glucose medium. Similarly, injection of Txnip shRNAs intravitreally leads to less mitophagy in Muller glia in a diabetic model. More work clearly needs to be done to investigate how Txnip might provide benefits to cone mitochondria, including investigation of the role of thioredoxins, Txnip, and mitochondrial biogenesis or mitophagy.

Reviewer #1: (Recommendations for the authors)

1. Injections of the Txnip gene from AAV with cone-specific promoters slows retinal degeneration. A control that is missing throughout this very interesting and significant report is direct evidence that Txnip actually is being overexpressed. My confidence in the conclusions would be stronger if the authors could show by immunoblot or by IHC that there is normally some endogenous expression of Txnip in cones and that when the eyes have in them AAV carrying a cone-specific promoter and the coding sequence for Txnip that Txnip expression in cones increases substantially. It would be very helpful if the level of over-expression could be quantified.

Please refer to our response to the Editor (point #1) above. The relevant data and additional data can be found in Author response image 1 for Txnip IHC at the end of this letter, a new Figure 5—source data 3 for RNA-seq raw reads, and Figure 5—figure supplement 1B (originally Extended Data Figure 5b) for ddPCR.

2. In theory when Txnip is over-expressed in cones there should be a decrease in GLUT1 expression in cones, but not in RPE cells. This report would be stronger if the authors could show by IHC that cone expression of GLIT1 is diminished in the Txnip cone OE retinas but GLUT1 would not be diminshed in the cones that express the mutant Txnip that is incapable of stimluating endocytosis of GLUT1.

Please refer to our response to Editor (point #1) above. The relevant data and additional data can be found in Author response image 2 for GLUT1 IHC at the end of this letter, and a new Figure 5—source data 4 for RNA-seq raw reads.

3. Lines 259-261. The finding that expression of ldhb prolongs survival of cones in these and other experiments throughout the report is interesting. It implies that cones are using lactate. But I would expect when rods are gone there is not much lactate in the retina. Please check that – i.e. measure lactate levels in the WT and rd1 retinas. The assay could be done with a commercial kit. If the lactate levels are low in the rd1 retina then it is puzzling why enhanced ldhb expression prolongs survival. Please address this either here or in the discussion. (Also, it would be helpful to confirm with an antibody that ldhb is being expressed (in the treated retinas at higher than normal levels).

Our data suggest that Txnip reprograms the cones via an unknown mechanism to use lactate more effectively than usual. This does not necessitate more Ldhb protein, but we too wondered if there might be more Ldhb protein in cones following Txnip overexpression. We tried LDHB antibodies from Sigma-Aldrich (SAB2108609) and Thermo Fisher Scientific (PA543141) for IHC, but neither worked well (i.e. low signal-to-noise ratio). This is the reason we created AAV8-RedO1.7-Ldhb-FLAG, and IHC stained against FLAG to test the effectiveness of the Ldhb shRNAs (Figure 3—figure supplement 1A, originally Extended Data Figure 3a). As we are not aware of any function of Ldhb beyond the conversion of lactate to pyruvate, our interpretation of the shRNA knockdown data is that lactate conversion to pyruvate via Ldhb, which is required for Txnip rescue. In support of this, overexpression of Ldha reduced the rescue by Txnip, which is consistent with the conversion of lactate to pyruvate as providing a benefit to cones (Figure 3—figure supplement 1D,E, originally Extended Data Figure 3d,e).Measurement of lactate levels in the retina would reveal the lactate level of the entire retina. As cones are a small fraction of total cells, and we do not know how much lactate is required or where, we do not think that measurements of total retinal lactate would be interpretable. Interestingly, the lactate level is lower than the glucose level in mouse serum, yet the turnover rate of lactate is much higher than it is for glucose in circulation, to feed the TCA cycle of non-CNS organs e.g. muscle (Hui S, 2017, Nature). As our manipulation is specific to a small percentage of cells, it would be challenging to measure the lactate consumption in Txnip treated cones.

4. I think readers would like to know what happens to the phosphorylation state of mTOR in cones in these experiments and I think it would be relatively simple to test. In some cells phosphorylation of mTOR in the absence of glucose can be harmful, I think because it is prepping cells for a metabolic state that does not exist in the tissue. The Cepko lab reported previously that mTOR phosphorylation in cones is diminished in rd mutants, consistent with a compromised supply of glucose to the retina. What effect does Txnip expression in cones have on mTOR phosphorylation (diminish, increase or no effect) and what effect does combined over-expression of HK1 and PFK1 expression have on mTOR phosphorylation?

We would also like to know, but we have not been able to find an antibody that shows the mTOR phosphorylation state since our original publication of this observation. The antibody that we used in that study no longer exists, and though we and Claudio Punzo (who originally did that work) have tried to find another one that works, we have been unable to do so.

5. Lines 277-286. Please confirm by IHC or immunoblot that the wt or dnHIF1 is over-expressed and, if possible, quantify by how much it is over-expressed.

We tried one HIF1A antibody from Novus Biologicals (NB100-479), and found a high background signal for IHC in the RP retina. In our previous study (Punzo et al., 2009), we found that RP cones showed stabilized HIF1A. Even if we had a suitable antibody, this signal might mask overexpression of Hif1a or dnHIF1A.

6. Extended data 6a and lines 300-302. I would have expected GLUT1 to be on the basal and apical surfaces of the Txnip.CS.LLAA mutant overexpressor but it seems to be only on the apical surface. Please mention and discuss this.

The “bright band” of GLUT1 is actually on the basal surface, not apical. We have updated the figure and legend to indicate this. With Best1-Txnip addition, the apical surface of the RPE collapses onto the photoreceptors, which also have GLUT1. These structural changes make it hard to tell how much GLUT1 is present on the apical surface of the RPE, when they express the CS.LLAA mutant compared to the WT Txnip.

Reviewer #2 (Recommendations for the authors):

The manuscript includes extensive data. However, several points require clarification and the text and figures need consistency. Specific comments/suggestions are as follows:

The authors concluded previously that cone starvation and degeneration are due to shortage of glucose in four animal models of RP (Punzo et al., 2009). However, except rho-/-, which is not the focus in this manuscript, the rest three animal models were not used in this study. It is unclear whether rd1 and rd10 also have shortage of ATP and how severe the shortage is as compared to the WT, or it is simply more ATP can better maintain cone survival.

We did not use Pde6γ-/- (as we did in Punzo et al. 2009), because its rod-cone degeneration rate is similar to that of Pde6β-/-, i.e. the rd1 strain. We also did not use the transgenic P23H strain (as in Punzo et al.) or a more recent P23H knock-in strain from the Palczewski Lab, due to the extremely slow rod-cone degeneration rate. We used the rd10 strain, which carries a Pde6β hypomorphic mutation instead of a null mutation. The rod-cone degeneration speed of rd10 is intermediate between those of rd1 and Rho-/- (Figure 1—figure supplement 1A, originally Extended Data Figure 1a), thus allowing us to investigate mutant strains with 3 rates of degeneration. Given that all of these mutant strains have a rod-specific mutation, and cone degeneration is secondary, our hypothesis throughout all of this work is that we are dealing with a non-autonomous set of problems for genetically wildtype cones. In Punzo et al. 2009, as well as several other gene therapy publications from our lab (Xiong et al. 2015, Wang et al. 2019, 2020, Wu et al. 2021, Chinchore et al. 2019), any treatment that benefits rd1 also benefits the rd10 and Rho-/- strains. Also, as we showed in Punzo et al., the changes in cones across the original 4 strains that we used had the same hallmarks, which suggests that the mechanisms leading to cone death are common across the strains. The additional time and expense of carrying out the Txnip experiments on P23H did not seem worthwhile in light of these findings.

The authors claim the treatment "correlates with the presence of a healthier mitochondria". As shown in Figure 5a, although the size of mitochondria was bigger in the treated groups compared to the control, those mitochondria did not seem to harbor a typical morphology. More transmission electron microscopy (TEM) images of different magnitudes are needed to show the localization of these mitochondria as well as the morphology of more mitochondria. Without the images from WT, it is hard to evaluate whether it is due to strain of the mice or other factors, or how a healthy mitochondrion should look like. Likewise, a much higher membrane potential in mitochondria does not necessarily mean healthier mitochondria. Additionally, the authors should show the mitochondria morphology of P50 rd1 as well, as that's the end point of the whole experiments and can provide useful information on the duration of the treatment.

Please see our response to the Editor (point #2) above. We have supplied more TEM images to address the question of mitochondrial morphology (Figure 5—figure supplement 2B-D). We are not claiming that Txnip-treated RP cones, or their mitochondria, look like wild type, only that they show several features of mitochondrial morphology and function that correlate with greater health. The TEM data show that they are larger in cross-section, the mitoRFP shows that they have a greater membrane potential and appear more “numerous”, the JC-1 dye aggregation shows greater membrane potential. All of these assays are standard assays used to investigate mitochondrial status. Furthermore, PercevalHR imaging showed that there is more ATP generated by Txnip treated cones, which is most likely due to better mitochondrial function, and that this is true when lactate is supplied, which is dependent upon Ldhb. In keeping with these data, Txnip C247S, provided for more cone survival, as well as had correlated benefits to cone mitochondria. Our RP cone RNA-seq data showed more ETC mRNAs, in correlation with the other assays of mitochondria. We did not rely only on TEM, but performed several independent assays to investigate the effects of Txnip on mitochondria.

Although the flat mount images in this manuscript show the overall cell survival in the whole retina, immunostaining of retinal sections is needed to address important questions related to retinal functions such as the structure of the retina, the morphology of the survival cone photoreceptors and the subtypes of the surviving cones, i.e., whether Txnip can maintain both S- and M- cones. The authors mentioned "this combination also preserved the RP cone outer segments" (line 312); yet, it is hard to evaluate the outer segment by flat mount, although a slight difference may be noticeable between the two groups.

We recently harvested and sectioned some P122 rd10 eyes transduced with RedOTxnip or RedO-Txnip + Best1-Nrf2. These sections are stained for S- or M- opsin separately. It showed that Txnip preserved expression of both S- and M- opsins, and Txnip + Best1-Nrf2 led to better cone outer segment structures with enriched S- and M- opsin (new Figure 8—figure supplement 1A). Again, we do not claim wildtype cone outer segment morphology, only that the combination of RedO-Txnip + Best1-Nrf2 gave the best outer segment morphology of degenerating cones that we have seen.

The automated method for cone quantification (1/2 radius cell count) that counts H2BGFP-positive cones in the central half of the retina (previous calculation of the diameter of flat mount retinas) raises some concerns. Is the area of the retina in the 1/2 radius the same between samples? Not all flat-mounted retinas look the same (differences due to retina dissection), and this can influence the area used to count cones.

When we began studies of RP cone rescue, and needed to quantify cones, we used multiple methods of IHC for cone markers (PNA, anti-opsin, anti-cone arrestin, cone lacZ transgene, cone RNA quantification). Due to lack of good morphology and a reduction in expression of cone genes in sick cones, we found all of these methods to be difficult and the IHC methods to be subjective. However, the H2BGFP protein seems to be quite stable, as predicted if it is localized within chromatin which is not turning over at an appreciable rate in cones. The fact that it shows bright nuclear staining made quantification, by eye or by an automated program, to be the most robust method that we have used. The specificity of expression by the RedO promoter, after rods are gone (some of which can express at a low level), made it possible to count from the flat mount. We bench marked this method to FACS quantification using anti-cone arrestin (see Wang et al. 2020). In addition, we provide data here showing the correlation with FACS quantification (new Figure 1—figure supplement 2C).

However, to address the point of variability in dissections and flat mounting, each retina is outlined and then the approximately central point of the optic nerve, which is unambiguous, is used to draw the central 50%. We have not shown the best images in our figures, but have shown typical images. The dissections and flat mounting procedure are quite reproducible as we have become quite proficient with these methods. There is certainly some natural variation of area among the samples, which is why we use the ½ radius as a normalization of each retina’s area. In addition, please note that the number of retinas for each experiment is quite high, and we include controls from the same animal (e.g. one eye injected with control and the other eye with Txnip). The P50 FVB rd1 control cone count is usually consistently around 4,000 in the central region using this method.

In addition, is this method counting all the cones present in the sample? Are all cones H2BGFP-positive? What is the viral transduction efficiency (alone and co-injecting two AAVs)? Is it the same between samples? Are authors using z-stack confocal images for quantification?

The injection of 2.5 x 108 vg/eye RedO-H2BGFP labels 20% of cones by our own estimation using WT mouse cone counts from Jeon, Strettoi and Masland (1998, J Neurosci). We have added this information in the Results section, where we first describe this labeling.

Because there is no difference between control and Txnip in P20 rd1 cone counting (Figure 1B, and new Figure 2—Figure supplement 1C), we do not think there is any difference in H2BGFP transduction rate alone vs. co-injection. We do not believe that we need to label every cone, but to reproducibly sample the existing cone population. It is also important to note that we may miss rescued cones with this method, when a cone is infected with Txnip but not H2BGFP. This is a loss that we are willing to take, as it deflates, rather than inflates, the rescue. The trade-off in the ease and confidence in scoring the H2BGFP nuclei is one we are quite willing to make.

As described in Methods-histology, we used a commercial fluorescence microscope with a 10x objective to capture and stich the images. By focusing on the residual ONL, which is only one cell layer thick, we do not need z-stacks to capture all the H2BGFP labeled cones in the RP retina. Overall, we believe the RedOpsin-H2BGFP counting method could become a useful method to assay RP cone survival for the field. We have deposited our MATLAB code for cone quantification on Github (https://github.com/sawyerxue/RP-cone-count), and it will be released to the public soon.

It is appropriate to quantify density of cones (number of cones/mm2) in defined areas of interest, which should be the same for all the samples. To achieve this, a proper orientation of the whole-mounted retina is necessary, and this information is lacking in the images included in the manuscript.

By using a computer program to count all of the labeled cones within a ½ radius circle of the entire retina, we were able to screen many vectors and many retinas, which alleviates concerns of measuring cone rescue only in a specific, perhaps selected or biased, area. In another study in our lab, we are quantifying the survival of peripheral cones, where we do see a bias in survival of the dorsal cones. In the current study, by counting all of the cones in the central retina and using a fairly large sample size for all studies, we do not see the need to orient the retina. The reproducible number of cones in the central retina in the control group (approximately 4,000) again suggests that random orientations do not create biases or irreproducible counts.

The authors could consider the quantification of "holes" or "craters" in the cone mosaic as an indicator of differential treatment effect.

As described in the text, craters are far more common in the FVB strain of the rd1 mutant. We assayed craters in 4 other inbred strains of mice that carry the rd1 allele and saw very few craters (Punzo and Cepko, unpublished). The cone counts do not correlate with the amount of these craters. Interestingly, we found that treatment of the RPE with AAV-Best1Nrf2 almost completely eliminated the FVB craters (Wu et al. 2021), without much benefit to cones. This suggests that the craters originate with problems in the RPE (oxidative damage, inflammation, perhaps metabolic problems) as Nrf2 may affect any or all of these problems. The lack of a benefit to cones is interesting, as we would have predicted an effect.

The authors do not quantify the level of Txnip expression in transduced retinas. It would be important to know the level of overexpression they have achieved and compare it with the expression in untreated RP retinas. In addition, it would be relevant to know if treatment effects correlate with Txnip expression levels.

Please see our response to the Editors (point #1) above on the Txnip antibody issues for IHC. The RNA level of Txnip treated vs. control cones can be found in a new Figure 5— source data 3. Due to the fact that cones normally express almost no Txnip mRNA, the increase of Txnip mRNA level is huge when adding RedO-Txnip.

OMR (based on head movements of mice observed by a human observer) can't be directly equated to visual acuity (see PLoS ONE 8(11): e78058, 2013; J Neurophysiol 118: 300-316, 2017). One needs to interpret such data with caution. Is it possible to do multi-focal ERG?

We agree with the reviewer that the OMR is sometimes not equal to visual acuity, because OMR can be used to measure different visual performances. However, visual acuity is one of them, and our use of it here did measure this parameter. A common visual performance that can be measured by OMR is contrast sensitivity, which we did not test here. By definition, visual acuity is equal to “spatial frequency threshold”, which is exactly the visual performance that we measured using OMR in this and previous studies (Xiong W et al., 2015; Wang SK et al., 2019, 2020). Based on these studies, we believe that the visual acuity measured by OMR reflects areas of improved cone function, which may reflect increased cone density and health in e.g. central retina.

We are not sure about the relevance of the two references provided by the Reviewer here, because both references support OMR as a reliable method of quantifying visual performance, including acuity. In PLoS ONE 8(11): e78058, 2013:

“Exemplary, we show that automatically measured visual response curves of mice match the results obtained by a human observer very well. The spatial acuity thresholds yielded by the automatic analysis are also consistent with the human observer approach and with published results. Hence, OMR-arena provides an affordable, convenient and objective way to measure mouse visual performance.”

J Neurophysiol 118: 300-316, 2017:

“We provide the first direct comparison of OMR and OKR gains (head or eye velocity/stimulus velocity) and find that the two reflexes have comparable dependencies on stimulus luminance, contrast, spatial frequency, and velocity.”

Multi-focal ERG is for animals with large eyes, including humans. For mice, which have tiny eyes, the Reviewer might be referring to focal ERG instead. Since we have the entire retina infected with P0 subretinal injection, i.e. not a partial infection as in human subretinal injections, we do not see the advantage of using focal ERG vs. full-field ERG. We observed no difference between control vs. Txnip in full-field ERG in the P40 rd10 mice (see new Figure 1—figure supplement 2E). Because ERG reflects overall phototransduction across the entire retina (Xue Y… Kefalov VJ, 2015a,b, 2017, 2020), but not improved functional cone density, i.e. improved visual acuity where cone survival is improved in the center, this result of the ERG does not argue against the positive OMR visual acuity results. Because cone loss is greatest in the central retina at the ages measured, the OMR result likely supplies the most robust measure of an improvement. In addition, this ERG result at least suggests that Txnip does not make the RP cone phototransduction worse than the control, which is an important consideration for therapeutic applications.

The authors confirmed the effect of Txnip on cone survival using three different animal models. However, in some follow-up experiments, the authors used these models interchangeably and it leads to confusion. For example, while rd1 is the main focus of the manuscript, the authors did not show the rescued visual acuity of these mice. Even if there is no effect of Txnip on visual acuity in these mice, the authors should still mention this to provide some information of the effectiveness of the treatment on some fast-degenerating mouse models. In addition, the authors used rd10 and rho-/- as well in the manuscript. Did the authors test the mechanisms they found using rd1 on rd10 and rho-/-? These data would be helpful to discriminate whether the switch of energy source by Txnip in the survival of cone cells is specific to rd1.

We added a sentence in Methods: “Optomotor tests were conducted on rd10 and Rho-/- mice, but not with rd1 strain, which loses vision at a very early age before any meaningful test can be performed.”

We have done RNA-seq with P90 Rho-/- mice treated with Txnip vs. control (Figure 5— figure supplement 1A, originally Extended Data Figure 5a), and found a shared upregulation of mitochondrial ETC gene upregulation with rd1 cones (Figure 5—source data 1, originally Supplementary Table 2). As explained above to Reviewer #2 (first point), cone degeneration shares a similar pattern across strains (Punzo et al., 2009). Given the improvement in rd10 and Rho-/- vision via the OMR assay, the RNA-seq results, and the common hallmarks of degeneration across all of the strains, we did not believe it necessary to perform more assays on the rd10 and Rho-/- strains. Rd1 was the preferred strain as it is more rapid than the other strains.

Figure 8a and 8b described the effect of vector combinations on cone survival in rd1, whereas Figure 8c actually mentioned the effect on outer segment in rd10. The authors did not even mention this switch of animal models in the text. The effect on outer segment in rd10 mice does not mean it would surely give the same effect in rd1 mice. It is unclear whether there was no improvement of outer segment in treated rd1 mice, or the vector combination did not have any effect on cone survival in rd10 mice.

The results from the combination treatment are quite new, and we were excited to include them here. However, they are not central to the overall finding that Txnip rescues cones in the 3 strains where we assayed cone survival (Figure 1), nor any of the other data on the mitochondria or Ldhb, or Parp1 KO. They are a result from our ongoing effort to combine treatments, as described above. But to address the question raised by the Reviewer, we have not examined the rd1 strain with anti-opsin with this combo, nor did we measure cone survival in P130 rd10 mice.

Reviewer #3 (Recommendations for the authors):

In order to strengthen this manuscript, the following suggestions should be addressed:

1. Did the authors observe the difference in mice with or without NNT deletion? Is there an NNT deletion in rd1, rd10, parp1-/-, and rho-/- mice?

We thank the reviewer for bringing this to our attention. The Nnt null mutation (MGI: 3626282) was observed only in the C57BL/6J background, and only rd10 mice in this study are on C57BL/6J background. We confirmed the presence of this mutation in our C57BL/6J and rd10 colonies by genotyping through Transnetyx, and found no nnt mutation in other strains (i.e. rd1, Rho-/-, Parp1-/-, CD1 or BALB/c). This information has been added in the Animals section of Methods.

Since rd10 mice are also responsive to Txnip rescue, NNT presence or absence does not affect the ability of Txnip to rescue. This result provides additional support that Txnip is a promising gene-agonistic therapy candidate for RP patients with various genetic backgrounds.

2. Extended Data Figure 1b and 1d: to show AAV8-RO1.7-GFP expression in cone photoreceptors, the Arr3 antibody will be better than PNA since PNA could only express in the cone outer and inner segments. Extended Data Figure 1b showed some GFP in the cytoplasm, is this the H2B-EGFP?

To reveal the full cone structure in different conditions, a new Figure 1—figure supplement 2A has been added with ARR3 staining. However, we believe PNA staining, which labels cone extracellular matrix including cone pedicles, is a more sensitive readout for toxicity to cones than ARR3 staining (Xiong, Wu, Xue et al., 2019, PNAS).

The GFP signal in Extended Data Figure 1b (now Figure 1—figure supplement 1B) is from the GFP-Txnip fusion protein, which does not have the H2B tag. We have updated all figures with in-panel color-code info to avoid any confusion to other readers.

3. Figure 1b: are these numbers within the 1/2 radius or within selected small boxes? If these numbers are from selected small boxes, how many small boxes from each were selected? The authors should explain how they selected the small boxes as there is some variation on the dorsal retina or ventral retina. For example, in P130 rd10, the cone cell densities are quite different in the four leaves of the inner circle. Same question for the P50 rd1, there is some area with a patchy absence of cone cells. If the numbers are within 1/2 radius, why use 1/2 radius rather than 2/3 or 1/3 radius?

The small boxes in Figure 1a were enlarged in Extended Data Figure 1c (now Figure 1—figure supplement 1C) to demonstrate the MATLAB program’s results of cone counting. Rather than just count the cones in the small boxes, Our MATLAB program counted all of the cones within the ½ radius of the entire retina (as a circle) to avoid any uneven degeneration in the four leaves.

To avoid any confusion, we have updated the Figure legend of Figure 1a to better explain the small boxes, which now reads: “All H2BGFP-labeled cones were counted within the central retina defined by the ½ radius (i.e. not just the cells from the small boxes).”

4. It would be helpful if the authors explained the reason behind each time point, i.e., P50 for rd1, P130 for rd10, and P150 for Rho -/-.

We have been using these ages as a standard for cone degeneration based on our previous assays of cone degeneration across time in the various strains (Punzo el al., 2009; Xiong et al., 2015; Wang SK et al. 2019, 2020). We have added an explanatory sentence in the text, and the progression over time is illustrated in Figure 1—figure supplement 1A.

5. Figure 5i: the number of cone cells at 1/2 radius in the whole mount retina looks quite similar, but it seems the number of cone cells in the periphery of control retina is more than the number in the +Ldhb retina.

As we have mentioned in Methods – Automated cone counting, the brightness in certain parts of the retina does not correlate with the number of cells. We believe our MATLAB quantification method produces the most reliable readout for the number of surviving cones. The central ½ radius is the best place to count, as the peripheral retina can have a portion of cones that live an extraordinary period of time, as we are attempting to understand. Moreover, the central retina is more critical for visual acuity than the peripheral retina.

6. Bracket "[]" in Figure 4, and curly bracket "{}" in Figure 5 indicate sample size. Are these the sample sizes of the image/retina or samples sizes of the mice?

This information is in the corresponding figure legends. Here briefly, in Figure 4b: “[]” is the number of images taken from regions of interest of multiple retinas. In Figure 5b: The number in the curly bracket “{ }” indicates the sample size, i.e. the number of mitochondria from multiple cones of >one retina for each condition. We duplicated this information to all other figures where it applies.

7. Figure 5: it is unclear how reliable the Mt size is, because the EM images are not SBF-SEM. The shape and morphology of mitochondria is in 3D and the EM images are taken from a different orientation and thus may have different results.

We agree with the reviewer that SBF-SEM is a better technology for mitochondria morphology. However, when we obtained the TEM imaging from multiple samples, it was very striking that the larger mitochondrial cross sections were in WT Txnip and Txnip.C247S treated rd1 cones rather than in controls, as reflected by the distribution of outliers in these group (Figure 5B). We have added a new Figure 5—figure supplement 2 showing more mitochondrial images from different conditions.

8. Did the authors observe any difference in the number of mitochondria in Txnip-treated retina?

As shown in the new Figure 5—figure supplement 2, we did not observe any obvious difference in mitochondria numbers (also see response to Reviewer #2). As the Reviewer pointed out, TEM might not be the best technology to answer this question. From our flatmount mitoRFP images (Figure 5—figure supplement 1C,D, originally Extended Data Figure 5c,d), there is an increase in mitoRFP density in the central retina by Txnip. This likely reflects an increase of mitochondrial density in this region.

9. Does Txnip increase mitochondrial biogenesis?

As explained in section# 3 to the Editors, please refer to the new Figure 5—figure supplement 2A and new Figure 5—source data 2 for RNA-seq data on Txnip treated WT cones.

10. It would be better if the authors could clarify the meaning of n.d. in "Wu et al., n.d.".

This work was previously in press, and recently published. We have updated this reference.

11. Line 336-337: it remains unclear what the role of anti-oxidative stress is in the Txnip-mediated cone rescue effect. It would be helpful if the authors added an experiment to demonstrate the changes in ROS in Txnip (wt and C237S)-mediated activities.

The Txnip WT allele is believed to decrease the anti-oxidative stress function of thioredoxin through binding that requires the Txnip C247 residue , i.e. the WT allele should increase oxidative stress. See the discussion above regarding the role of Txnip in oxidative stress. It is clear that more work needs to be done to understand our findings relative to oxidative stress.

12. Line 353: it would be helpful if the authors could explain why the Txnip rescue did not depend on ketolysis or ß-oxidation.

While we cannot know the answer to this question, we did speculate, in the original Discussion: “An important factor in the reliance on Ldhb could be the availability of lactate, which is highly available from serum (Hui et al., 2017). Lactate could be transported via the RPE and/or Müller glia, and/or the internal retinal vasculature which comes in closer proximity to cones after rod death. Ketones are usually only available during fasting, and lipids are hydrophobic molecules which are slow to be transported across the plasma membranes. Moreover, lipids are required to rebuild the membrane-rich outer segments, and thus might be somewhat limited.”

13. Line 487-488: "The GFP fluorescence intensity served as a fast 488 and direct read out of the knockdown efficiency of these shRNAs." The GFP after IRES can sometimes too weak to produce a fast and direct readout. Do the authors have the image/a figure on this?

Yes, these images were supplied in the original version, in Extended data Figure 8-11 (now Figure 2—figure supplement 2, Figure 3—figure supplement 2-4).

14. It is unclear whether the control group in this study refers to the non-injected eye or to saline injections

In Methods- subretinal injection section, we have added a sentence that reads: “All of the control groups in this study refer to AAV reporter (e.g. H2BGFP or PercevalHR) injection alone.”

https://doi.org/10.7554/eLife.66240.sa2

Article and author information

Author details

  1. Yunlu Xue

    1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    2. Department of Ophthalmology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2088-9826
  2. Sean K Wang

    1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    2. Department of Ophthalmology, Harvard Medical School, Boston, United States
    3. Howard Hughs Medical Institute, Chevy Chase, United States
    Contribution
    Resources, Formal analysis, Validation, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Parimal Rana

    Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    Contribution
    Formal analysis, Validation, Investigation
    Competing interests
    No competing interests declared
  4. Emma R West

    1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    2. Howard Hughs Medical Institute, Chevy Chase, United States
    Contribution
    Software, Formal analysis
    Competing interests
    No competing interests declared
  5. Christin M Hong

    1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    2. Howard Hughs Medical Institute, Chevy Chase, United States
    Contribution
    Formal analysis, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Helian Feng

    Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, United States
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  7. David M Wu

    1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    2. Department of Ophthalmology, Harvard Medical School, Boston, United States
    3. Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, United States
    Contribution
    Resources, Software, Writing - review and editing
    Competing interests
    No competing interests declared
  8. Constance L Cepko

    1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    2. Department of Ophthalmology, Harvard Medical School, Boston, United States
    3. Howard Hughs Medical Institute, Chevy Chase, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing
    For correspondence
    cepko@genetics.med.harvard.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9945-6387

Funding

National Eye Institute (K99EY030951)

  • Yunlu Xue

National Eye Institute (U01EY025497)

  • Constance L Cepko

Alcon Research Institute

  • Constance L Cepko

Astellas Pharmaceuticals

  • Constance L Cepko

Howard Hughes Medical Institute

  • Constance L Cepko

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Sui Wang, ChangHee Lee, Sylvain Lapan, Gabby Niconchuk, Brian Rabe, Cem Sengel, Sophia Zhao, Yuji Atsuta, Wenjun Xiong, Ryoji Amamoto, Grace Wallick, Gary Yellen, Zhongjie Fu, Zhengping Hu, Maryna Ivanchenko, Paula Montero-Llopis, Microscopy Resources on the North Quad, Maria Ericsson, Electron Microscopy Facility, Flow Cytometry of Immunology, Marcelo Cicconet, Image and Data Analysis Core of Harvard Medical School, Genomics Platform of Broad Institute, Metabolomics Core Resource Laboratory of New York University, and Frans Vinberg at University of Utah for discussions and technical support. We also thank Botond Roska, Jacob Keller, Loren Looger, Leah Byrne, John Flannery, William Hahn, David Root, Lewis Cantley, Matthew Vander Heiden, Geoff Wahl, Michael Davidson, Clark Distelhorst, Bong-Kiun Kaang, and Thorsten Mascher for plasmids. This work was funded by the National Institute of Health (K99EY030951 to YX and U01EY025497 to CLC), Alcon Research Institute (CLC), Astellas Pharmaceuticals (CLC), and Howard Hughes Medical Institute (CLC).

Ethics

Animal experimentation: All animal experiments were approved by the IACUC of Harvard University in accordance with institutional guidelines (protocol number: IS1695).

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Claude Desplan, New York University, United States

Reviewer

  1. James B Hurley, University of Washington, United States

Publication history

  1. Received: January 5, 2021
  2. Accepted: March 30, 2021
  3. Accepted Manuscript published: April 13, 2021 (version 1)
  4. Version of Record published: April 28, 2021 (version 2)

Copyright

© 2021, Xue et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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