Retinitis pigmentosa (RP) is an inherited retinal disease in which there is a loss of cone-mediated daylight vision. As there are >100 disease genes, our goal is to preserve cone vision in a disease gene-agnostic manner. Previously we showed that overexpressing TXNIP, an α-arrestin protein, prolonged cone vision in RP mouse models, using an AAV to express it only in cones. Here, we expressed different alleles of Txnip in the retinal pigmented epithelium (RPE), a support layer for cones. Our goal was to learn more of TXNIP’s structure-function relationships for cone survival, as well as determine the optimal cell type expression pattern for cone survival. The C-terminal half of TXNIP was found to be sufficient to remove GLUT1 from the cell surface, and improved RP cone survival, when expressed in the RPE, but not in cones. Knock-down of HSP90AB1, a TXNIP-interactor which regulates metabolism, improved the survival of cones alone and was additive for cone survival when combined with TXNIP. From these and other results, it is likely that TXNIP interacts with several proteins in the RPE to indirectly support cone survival, with some of these interactions different from those that lead to cone survival when expressed only in cones.
Yunlu Xue1 and Constance L. Cepko2 1Lingang Laboratory, Shanghai 200031, China 2Departments of Genetics and Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA Correspondence: ylxue{at}lglab.ac.cn; cepko{at}genetics.med.harvard.edu
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.
Pigment regeneration is critical for the function of cone photoreceptors in bright and rapidly-changing light conditions. This process is facilitated by the recently-characterized retina visual cycle, in which Müller cells recycle spent all-trans-retinol visual chromophore back to 11-cis-retinol. This 11-cis-retinol is oxidized selectively in cones to the 11-cis-retinal used for pigment regeneration. However, the enzyme responsible for the oxidation of 11-cis-retinol remains unknown. Here, we sought to determine whether retinol dehydrogenase 10 (RDH10), upregulated in rod/cone hybrid retinas and expressed abundantly in Müller cells, is the enzyme that drives this reaction. We created mice lacking RDH10 either in cone photoreceptors, Müller cells, or the entire retina. In vivo electroretinography and transretinal recordings revealed normal cone photoresponses in all RDH10-deficient mouse lines. Notably, their cone-driven dark adaptation both in vivo and in isolated retina was unaffected, indicating that RDH10 is not required for the function of the retina visual cycle. We also generated transgenic mice expressing RDH10 ectopically in rod cells. However, rod dark adaptation was unaffected by the expression of RDH10 and transgenic rods were unable to use cis-retinol for pigment regeneration. We conclude that RDH10 is not the dominant retina 11-cis-RDH, leaving its primary function in the retina unknown.
Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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., 2015; Figure 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 Download asset Open asset 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 Download 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 Download asset Open asset 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 Download asset Open asset 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 Download asset Open asset 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 deg
Abstract Continuous visual perception and the dark adaptation of vertebrate photoreceptors after bright light exposure require recycling of their visual chromophore through a series of reactions in the retinal pigmented epithelium (RPE visual cycle). Light-driven chromophore consumption by photoreceptors is greater in daytime vs. nighttime, suggesting that correspondingly higher activity of the visual cycle may be required. However, as rod photoreceptors are saturated in bright light, the continuous turnover of their chromophore by the visual cycle throughout the day would not contribute to vision. Whether the recycling of chromophore that drives rod dark adaptation is regulated by the circadian clock and light exposure is unknown. Here, we demonstrate that mouse rod dark adaptation is slower during the day or after light pre-exposure. This surprising daytime suppression of the RPE visual cycle was accompanied by light-driven reduction in expression of Rpe65 , a key enzyme of the RPE visual cycle. Notably, only rods in melatonin-proficient mice were affected by this daily visual cycle modulation. Our results demonstrate that the circadian clock and light exposure regulate the recycling of chromophore in the RPE visual cycle. This daily melatonin-driven modulation of rod dark adaptation could potentially protect the retina from light-induced damage during the day.
Abstract Vision is initiated by the reception of light by photoreceptors and subsequent processing via downstream retinal neurons. Proper cellular organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of Fat3 mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. The role of FAT3 in the response to high temporal frequency flashes depends upon its ability to transduce an intracellular signal. Mechanistically, FAT3 binds to the synaptic protein PTPσ, intracellularly, and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. These findings expand the repertoire of FAT3’s functions and reveal its importance in bipolar cells for high frequency light response.
Lamin B1 and lamin B2 are essential building blocks of the nuclear lamina, a filamentous meshwork lining the nucleoplasmic side of the inner nuclear membrane. Deficiencies in lamin B1 and lamin B2 impair neurodevelopment, but distinct functions for the two proteins in the development and homeostasis of the CNS have been elusive. Here we show that embryonic depletion of lamin B1 in retinal progenitors and postmitotic neurons affects nuclear integrity, leads to the collapse of the laminB2 meshwork, impairs neuronal survival, and markedly reduces the cellularity of adult retinas. In stark contrast, a deficiency of lamin B2 in the embryonic retina has no obvious effect on lamin B1 localization or nuclear integrity in embryonic retinas, suggesting that lamin B1, but not lamin B2, is strictly required for nucleokinesis during embryonic neurogenesis. However, the absence of lamin B2 prevents proper lamination of adult retinal neurons, impairs synaptogenesis, and reduces cone photoreceptor survival. We also show that lamin B1 and lamin B2 are extremely long-lived proteins in rod and cone photoreceptors. OF interest, a complete absence of both proteins during postnatal life has little or no effect on the survival and function of cone photoreceptors.
