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.

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).

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., 2020; Figure 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.

Figure 9

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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.

Animals rd1 mice were the albino FVB strain, which carries the Pde6b^rd1 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).

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

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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

   "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

   "This ORCID iD identifies the author of this article:" 0000-0002-9945-6387

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