Combination of blockade of endothelin signalling and compensation of IGF1 expression confers protective effects on degenerating retina

doi:10.21203/rs.3.rs-3194107/v1

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Combination of blockade of endothelin signalling and compensation of IGF1 expression confers protective effects on degenerating retina

https://doi.org/10.21203/rs.3.rs-3194107/v1

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published 22 Jan, 2024

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Retinitis pigmentosa (RP) and macular dystrophy (MD) cause severe retinal dysfunction, from which 1 in 4,000 people suffer worldwide. This disease is currently assumed to be intractable because effective therapeutic methods have not been established, regardless of genetic or sporadic traits.

Here, we took advantage of RP model mice in which the Prominin-1 (Prom1) gene is deficient and investigated the molecular events occurring at the outset of retinal dysfunction. We extracted the Prom1-deficient retina subjected to light exposure for a short time, carried out single-cell expression profiling, and compared the gene expression with that without stimuli. We identified the cells and genes whose expression levels change directly in response to light stimuli. Among the genes altered by light stimulation, we found that Igf1 was decreased in rod photoreceptor cells and astrocytes under light-stimulated conditions. Consistently, the insulin-like growth factor (IGF) signal was weakened in the light-stimulated photoreceptor cells. The recovery of Igf1expression with the adeno-associated virus (AAV) prevented photoreceptor cell death, and its treatment in combination with the endothelin receptor antagonist led to the blockade of abnormal glial activation and the promotion of glycolysis, and thereby resulted in the improvement of retinal functions, as assayed by electroretinography. We additionally demonstrated that the attenuation of mammalian/mechanistic target of rapamycin (mTOR), which mediates IGF signalling, leads to complications in retinal homeostasis. Together, we propose that combinatorial manipulation of distinct mechanisms is useful for the maintenance of the retinal condition.

Retinitis pigmentosa

Prominin-1

insulin-like growth factor (IGF)

gliosis

mammalian/mechanical target of rapamycin (mTOR)

adeno-associated virus (AAV)

single-cell RNA sequencing (scRNA-seq)

The daily information from the periphery of bodies is acquired mainly through visual transduction, and the loss of visual function causes severe impacts on the quality of individual lives. Understanding the initiation and progression of retinal degeneration and implementing therapeutic methods are therefore of great interest (1).

Among a number of eye diseases, RP and MD are major disorders occurring in the retina. In most cases, degenerations start in photoreceptors and/or retinal pigment epithelium (RPE) cells (1, 2), and the degeneration gradually spreads to the entire retina. One in 4,000 people suffers from RP and MD (3–5), and half of their cases are of genetic traits, and disorders are inherited from superior to inferior generations in specific pedigrees. More than 60 genes have been identified as causative for RP and MD, and Prominin-1 (Prom1/CD133), on which we focus in this study, is one such gene (6–10).

The Prom1 gene encodes a pentaspan membrane protein and is highly expressed in the retina (8), testis and kidney at the tissue level (11). Regarding subcellular localisation, PROM1 is present in the disc region of photoreceptor cells and in the microvilli of the cell membrane (12). The low similarity of the polypeptide sequence of Prom1 to other proteins makes it difficult to predict its physiological function. Nevertheless, PROM1 has been suggested to be involved in membrane functions, such as cholesterol localisation (13) and the formation of extensions (14) and extracellular vesicles (12, 13, 15). Thus, Prom1 is highly likely to be involved in membrane morphogenesis and vesicle secretion.

Mutations in the Prom1 gene have been reported in some families where people suffer from RP and MD (8). The mice attenuated with the Prom1 gene (Prom1-/-; Prom1-KO) well recapitulate the symptoms found in human patients, making the Prom1 mutant mice a suitable animal model to investigate the progression of retinal degeneration (9, 10). Prom1 mutant mice can be born normally; however, the retinas of the mutant mice start to degenerate immediately after the eyes open (9). Prom1-mutant mice exhibit severe photoreceptor loss and failure of autophagy of the RPE cells (16); therefore, drusen, the accumulation of lipids and proteins that should have been removed, accumulate around the RPE cell layer. Light stimulation is a major trigger of the retinal phenotype (6); Prom1-KO mice exhibit programmed cell death and progressive thinning of the outer nuclear layer (ONL) of the retina (6).

In our previous study, we conducted high-throughput expression profiling and identified genes whose expression levels are altered in the retina at the earliest step of retinal degeneration (6, 17). Although no significant change was found in any genes, except for Prom1 itself, in the 2-week-old Prom1-KO retina compared to the wild-type, more than one thousand genes were altered in their expression compared to the wild-type at 3 weeks after birth. This analysis showed that endothelin signalling was markedly activated in the Prom1-KO retina, which induced glial fibrillary acidic protein (Gfap) gene expression to activate glial cells. Although the activation of glial cells leads to the production of neurotrophic factors that may act to rescue photoreceptor cell survival and function (18–20), excess glial activation leads to gliosis, where glial cells clump together and become a spatial obstacle in the retina. In addition, the endothelin signal induces the stenosis of the retinal vessels by vasoconstriction (21), and the substance supply from the blood to the surrounding cells, particularly to the photoreceptor cells, becomes lacking. Treatment with antagonists targeting endothelin receptors can alleviate the phenotype, suggesting that excess endothelin signalling negatively affects the retina, and blockade of the endothelin signal partially recovers the phenotypes during retinal degeneration. The activation of the endothelin signal and subsequent gliosis has also been shown in other mutant mouse lines causing RP (22). Moreover, the results suggest that photoreceptor cell death does not occur cell-autonomously but proceeds through cell‒cell interactions.

However, several questions remain unsolved. For instance, unidentified signals other than those induced by endothelin appear to be involved in the progression of retinal degeneration. In addition, the details of cell‒cell communication during the onset of retinal degeneration have not been elucidated. Likewise, although a number of genes were found to be altered in their expression in the expression analysis, as the retina is composed of a number of different cells, the cells in which gene expression levels are altered cannot be identified as long as the analysis is performed in the whole retina.

As whole tissue analysis has resolution limitations, we now took advantage of single-cell expression profiling. We extracted RNA samples from two conditions of the retina of both Prom1-KO backgrounds, one with and the other without light exposure, and gene expression analysis was performed at the single-cell level. As a result, we found that IGF signalling was downregulated in photoreceptor cells and astrocytes. The combinatorial treatment of the inhibitor of the aberrantly upregulated signal and the compensation of the signal lacking in the mutant retina further and more efficiently recover the retinal phenotypes. We will further demonstrate that mTOR, which mediates IGF signalling, plays an essential role in photoreceptor survival and retinal tissue integrity.

The 11-day-old retina expresses retinal genes, and the Prom1 mutant is vulnerable to light stimulation

We first investigated expression in 11-day-old Prom1+/- retinas to ask whether functional cells exist in the retina early after birth. At this age, mice have not opened their eyes, and the retina has never been exposed to any stimulation from the periphery, including light exposure; therefore, the cells have not undergone environmental cues.

By immunostaining, we found rhodopsin (RHO) (Fig. 1A) and m-Opsin (Fig. 1B) signals, identifying outer segments of rod and cone photoreceptor cells, respectively, outside ONL. In addition, Nestin (Fig. 1C) and GFAP (Fig. 1D), marking Müller glial cells, were detected, as well as Prom1, as detected by b-galactosidase staining (Fig. 1E). Therefore, the functional cells have developed before the eyes open.

We next examined whether these cells also exist in the homozygotic mutants of Prom1. The retina was overall indistinguishable from the heterozygotic mutant (Fig. 1A-E), and the distribution of the proteins above was unchanged (Fig. 1F-J), suggesting that retinal development was unaffected by the loss of Prom1 function.

In our previous study (6), we demonstrated that retinas devoid of the Prom1 gene are highly susceptible to light stimuli, leading to aberrant activation of glial cells called gliosis. To determine whether early postnatal retinal cells respond to light exposure as in the juvenile and adult stages, we forcibly opened the eyes of the Prom1 heterozygotic and homozygotic mice, exposed them to 15,000 lux light stimulation for 3 hours, and returned them to the dark environment overnight to ensure the accumulation of mRNAs. The whole retina was then extracted, and the gene expression was compared with those reared in the continuous dark environment by reverse-transcription and quantitative polymerase chain reaction (RT‒qPCR).

As a result, we found upregulated gene expression of Endothelin-2 (Edn2), encoding the endothelin peptide (ET-2) that negatively affects retinal homeostasis (6, 17), and B-Cell Lymphoma 3 (Bcl3), which is involved in cell survival, under the light condition in the Prom1-/- retina but not in the Prom1+/- retina under either light or dark conditions (Fig. 1K). In addition, when the mice were reared in the dark until 3 weeks of age after temporal light exposure as stated above, GFAP signals in Müller glial cells extending into the ONL were detectable in the Prom1-/- but not in the Prom1+/- retina (Fig. 1L, M). Thus, glial cells are also reactive to the signals induced by light exposure.

Together, the results suggest that retinal tissue robustness against light-induced stress depends on the existence of Prom1 and that the Prom1-/- retina is vulnerable to light stimulation even before the mouse eyes open.

Single-cell expression profiling identified the cells and genes whose expression was altered by light exposure

We next sought to identify the genes and cells directly affected by light stimulation. For this purpose, we conducted single-cell gene expression profiling on retinas that were exposed to light stimulation. To minimise possible individual differences caused by the rearing environment, we employed two Prom1-KO mice that were born from the same mother and processed them in the same way as in the previous RT‒qPCR analysis (Fig. 1K). The retinal cells were then dissociated, and ten thousand cells were subjected to single-cell RNA sequencing (scRNA-seq) to compare the gene expression with another Prom1-/- mouse reared in persistent dark conditions (see Material and Methods for details).

From two sets of transcriptome data produced from both the dark and light conditions, the whole tendency of the gene expression was visualised by t-distributed stochastic neighbor embedding (t-SNE) expansion (Fig. 2A, Supplementary Fig. S1A). Overlaying spots did not exhibit apparent changes in the distributions of the cells, suggesting that the two samples were comparable in developmental and maturing conditions (Supplementary Fig. 1A).

Next, the cell types were categorised into 11 groups by the genes specifically expressed in each cell type (Table 1, Supplementary Fig. 1B). For instance, rod cells were sorted by the unique expression of RHO, Sag, Pdc, Gngt1, and Rp1 (55.3% in the dark and 59.8% in the light), and cone cells were sorted by Opn1sw (8.6% in the dark and 7.9% in the light). The other types of retinal cells were likewise identified by the genes expressed uniquely in the indicated cells (Supplementary Fig. S1B). The ranking of the population in the retina was unchanged overall between the two conditions (Table 1), suggesting that cell death or selective exclusion of specific cell types had not yet started.

In the rod photoreceptor cells, 271 genes (145 genes upregulated and 126 genes downregulated) were changed in their expression with p values lower than 0.05 (Table 1). This gene list included Edn2 as an upregulated gene, confirming that the endothelin peptide ET-2 is produced and emanates from rod photoreceptor cells (6, 17) (Fig. 2B). Moreover, the Gfap and Serpina3n genes, whose expression is responsive to ET-2 (23), were enriched in Müller glia and astrocytes by light stimulation (Fig. 2B, C), suggesting that the glial reaction had already started (24). In contrast, no genes were found to be altered in RPE cells (Table 1). This observation suggests that photoreceptor cells, rather than RPE cells, were the earliest cell type affected by light stimulation at the onset of RP.

We further found that bipolar and rod photoreceptor cells exhibited the upregulation of S100a8 and S100a9 (Fig. 2C), encoding calcium- and zinc-binding proteins activated upon neuroinflammation (25). We also found that a number of crystallin genes (Cry) were upregulated, including a-crystallin (Crya), whose expression has been shown to be upregulated by stress (26), under light conditions in most retinal cell types (Fig. 2C). Thus, the Prom1-/- retinal cells exhibit the stress response to light stimulation by inducing different sets of genes, and the response occurs not only in the photoreceptor cells where Prom1 is mainly expressed (Fig. 1E) but also in the surrounding cells.

We were also aware that some of the genes essential for visual functions were downregulated by light stimulation in the Prom1-/- retina. In photoreceptor cells, Arrestin3 (Arr3; rod and cone) and G Protein Subunit Alpha Transducin 1 (Gnat1; cone), both of which play critical roles in phototransduction, decreased (Fig. 2C, D), suggesting that Prom1 forms a gene regulatory network with the other genes for retinal functions and is an upstream gene.

Overall, single-cell expression profiling successfully identified the cells and genes that respond to light stimulation at the earliest stage of retinal degeneration.

Igf1 expression in rod photoreceptors and astrocytes is downregulated in the light-stimulated Prom1 -mutant and affects ribosomal protein S6 phosphorylation

We sought to identify extracellular molecules with altered expression, as changes in their expression levels may affect surrounding cells and regulate the condition of the entire retina. Along this line, we particularly became interested in Igf1, as Igf1 was found to be downregulated upon light stimulation in rod photoreceptors and astrocytes, whose characteristics are presumably similar to those of the surrounding cells (Fig. 2D; filled and open arrowheads). IGF1, encoded by Igf1, has been shown to have a neurotrophic effect on neurons, including photoreceptor cells, activating the pro-survival signal and anti-apoptotic pathways (27, 28). Consistently, a lack of IGF signalling by knocking out the IGF1 receptor gene leads to photoreceptor degeneration (29). Therefore, we speculated that the decrease in IGF1 is one of the triggers for photoreceptor degeneration.

Given that the IGF signal can be mediated by mTOR activation (30), we first sought to address the activation of the downstream protein. For this purpose, we asked if the ribosomal protein S6 is phosphorylated at Ser240 and Ser244 (hereafter denoted as pS6) because these residues are directly phosphorylated by the S6 kinase p70S6K via the activation of the IGF/mTOR signal (31). In the Prom1 heterozygotic littermate at 12 days old, pS6 was detected in the ganglion cell layer (GCL) and at border areas of the inner nuclear layer (INL) (Fig. 3A). At 3 weeks, the expression at the outer part of the INL became evident, and notably, the signals immediately outside of the ONL were found, in addition to the same cells at 12 days (arrowheads in Fig. 3B, B'; compare with the area indicated with open arrowheads in Fig. 3A). Moreover, pS6 is localised to the inner segment of the photoreceptor cells, as the signal was found to be complementary to that of RHO and s-Opsin localised to the outer segment (Supplementary Fig. S2). The pS6 signal at the inner segment of the photoreceptor cells was found even in the retina where the mice were reared in the continuous dark environment at 3 weeks (Fig. 3C), suggesting that phosphorylation occurs in an age-dependent manner but is independently regulated from the dark/light environment where the mice are reared.

In the Prom1-/- retina, pS6 was localised in areas similar to those of Prom1+/- at 12 days old (Fig. 3D); phosphorylation was found in the GCL and inner INL. In contrast, at 3 weeks old, while the localisation in the GCL and INL was the same as that in the Prom1+/- mice, significantly less pS6 was detected outside the ONL (Fig. 3E, E'; grey arrowheads). On the other hand, the presence of pS6 was observed in the Prom1-/- retina reared in the dark environment, as in the Prom1+/- (Fig. 3F). In these retinas, the number of ONL cells was comparable in both genotypes (Fig. 3G), suggesting that the decrease in pS6 was unlikely due to the secondary effect caused by the alteration in the number of photoreceptor cells. Therefore, light stimulation on the Prom1-/- retina caused a decrease in pS6 positivity at the inner segment of the photoreceptor cells.

To further address the correlation between IGF1 and S6 phosphorylation, we attempted to confirm that the S6 protein can be phosphorylated by IGF1. We therefore injected recombinant IGF1 protein into the vitreous body of 12-day-old wild-type mice and examined pS6 localisation in the retina. As a result, the injection of IGF1, but not control PBS, induced ectopic S6 phosphorylation, particularly in the photoreceptor and INL (Fig. 3H, I; white arrowheads). Thus, the cells are competent to respond to the IGF signal, and its downstream S6 protein can be phosphorylated.

Together, the data suggest that Igf1 is one of the downregulated genes upon light stimulation in the retina and is concomitant with the phosphorylation of the S6 protein.

Persistent IGF1 expression blocks photoreceptor cell death

We next asked if the compensation of the IGF signal in the Prom1-/- retina recovers retinal survival and functions. To ensure sustained IGF expression, we conducted adeno-associated virus (AAV) infection into the retina using intravitreal injection and investigated the ameliorating effects of the infection.

To validate AAV infection, we prepared 6-week Prom1+/- retinas that were either uninfected (Fig. 4A) or infected with AAV-Gfp at 2 weeks of age (Fig. 4B). We found that GFP signals were widely caused by infection in retinal cells (Fig. 4B).

Next, we infected AAVs conveying Gfp (control) or Igf1 into the Prom1+/- or Prom1-/- retinas with the same experimental schedule as above (Fig. 4B). In 6-week-old retinas, pS6 was localised at the GCL (Fig. 4C, C'; green arrowheads), INL (Fig. 4C, C'; yellow arrowheads), and photoreceptor layer (Fig. 4C, C'; orange arrowheads). While the signals were almost undetectable in the Prom1-/- retina upon the infection of AAV-Gfp (Fig. 4D, D'), the infection with AAV-Igf1 showed partially rescued signals of pS6 (Fig. 4E, E'), with some ectopic signals (Fig. 4E, E'; red arrowheads) detectable. Notably, a higher number of cells were found at the ONL in the Prom1-/- retina upon infection with AAV-Igf1 (brackets; Fig. 4D', E', F), suggesting that IGF signalling has a protective effect against retinal degeneration caused by a lack of Prom1 function.

We reasoned that the increased number of ONL cells was caused by reduced programmed cell death and conducted a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. We found several apoptotic cells in the Prom1-/- retina upon infection with control AAV-Gfp (Fig. 4H, H', J), which was never detected in the Prom1+/- retina (Fig. 4G, G', J) under the same experimental conditions; however, infection with AAV-Igf1 significantly decreased the number of TUNEL-positive cells (Fig. 4I, I', J). Therefore, programmed cell death was perturbed by the IGF signal.

We further investigated the effect of IGF1 on glial activation. A larger number of GFAP-positive glial cells reaching INL were detected in the Prom1-/- retinas than in the Prom1+/- retinas (Fig. 4K-L',N). However, this number did not decrease upon infection with AAV-Igf1 (Fig. 4M-N), suggesting that IGF signalling does not have an ameliorating effect on gliosis and further suggesting that IGF acts independently of neuroinflammation signals.

IGF/mTOR signalling has been shown to activate aerobic glycolysis, a metabolic process in which glucose is converted to pyruvate. Through several transfer reactions, the oxidised form of nicotinamide adenine dinucleotide (NAD⁺) is reduced to NADH, and adenosine triphosphate (ATP) is produced (32). Based on this knowledge, we asked if aerobic glycolysis was activated upon infection with AAV-Igf1 and measured the NADH/NAD⁺ ratio in 6-week-old retinas.

We found that the retinal cell extracts from Prom1-/- showed a lower ratio of NADH/NAD⁺ than those from Prom1+/- (Fig. 4O). In contrast, the AAV-Igf1-infected Prom1-/- retina exhibited a restoration of NADH/NAD⁺ levels, suggesting that the reduction reaction of NAD⁺ to NADH was activated by the overexpression of IGF1 (Fig. 4O).

Together, IGF1 has blocking activity against programmed cell death and thereby protects the retina, including mainly photoreceptor cells, without attenuating GFAP signalling.

The combination of IGF1 overexpression and blockade of endothelin signalling improves retinal function in Prom1-/- mice

Previous studies have shown that administering endothelin receptor antagonists ameliorates gliosis and suppresses photoreceptor cell death (6, 17). Therefore, we next attempted to understand the functional relevance of endothelin receptor antagonists and IGF on gliosis or retinal functions.

Herein, we used bosentan as the endothelin antagonist, which targets two endothelin receptors of EdnrA and EdnrB with a similar binding affinity (33–36) and is clinically used to treat pulmonary arterial hypertension (37). In this study, injections were performed intravitreally to ensure a local effect (Fig. 5A).

To validate the effect of bosentan, we first administered vehicle (control; dimethyl sulfoxide (DMSO)) or bosentan to 2-week-old Prom1+/- or Prom1-/- retinas. Vehicle or bosentan was additionally administered one week after the first injection (Fig. 5A for the injection schedule). The bosentan-administered Prom1-/- retinas showed significantly fewer GFAP-positive (Supplementary Fig. S3A-C') and TUNEL-positive cells (Supplementary Fig. S3D-F') than the control retinas treated with control DMSO. Therefore, bosentan is effective in blocking the excess activation of Müller glial cells and programmed cell death.

We next sought to address the correlation between the suppression of cell death and functional improvement and carried out electroretinography (ERG). In this physiological assay, two peaks of the initial negative (a-wave: reflecting the function of the photoreceptor cells) and the following positive peak (b-wave: mainly detecting the function of horizontal and Müller glia) evoked by a light pulse can be evaluated (Fig. 5B). Prom1+/- retinas showed comparable a- and b-wave amplitudes at 3, 5, and 8 weeks of age, suggesting that the 3-week-old retinas are already functionally similar to those of mature individuals (Fig. 5C, D). On the other hand, in the Prom1-/- retina, both a- and b-waves were significantly lower than those in the Prom1+/- retina at 3 weeks of age (Fig. 5C, D), confirming that retinal function had started to deteriorate before this stage, although the number of retinal cells was still comparable to that in the heterozygotic littermates. Furthermore, this response decreased over time in the Prom1-/- retinas, and the waveforms were almost undetectable at 8 weeks of age, suggesting that the Prom1-/- mice were completely blind at 8 weeks of age (Fig. 5B’-D).

Compared with the control AAV-Gfp treated Prom1-/- retina, the bosentan-administered mutants exhibited significantly improved the a-wave amplitude at 3 weeks of age. This trend persisted at 5 and 8 weeks of age (Fig. 5C, D; orange bars), and the b-wave amplitude was also found to be improved at 8 weeks of age, confirming that bosentan improves retinal function. Moreover, the combination of bosentan and AAV-Igf1 improved the ERG scores compared with the control group, and noticeably, all amplitudes of the a- and b-waves were significantly greater than those of the control Prom1-homozygotic mutants (Fig. 5B-D).

Therefore, while the single treatment with the endothelin signalling blocker on the degenerating retina exhibits the improvements of the retinal functions, the simultaneous manipulation of two signals, endothelin and IGF, exhibits superior protective effects.

mTOR-mediated signalling is required for photoreceptor survival

Among several intracellular downstream pathways induced by IGF (28, 38), we asked if the mTOR-mediated branch is involved in retinal homeostasis and tissue integrity. As the null mutation of the mTOR gene causes early embryonic lethality (39), we generated the drag-inducible and neural and glial- and temporal-specific knockout line (Nestin-CreERT2; mTOR f/f), where the mTOR gene was conditionally removed.

We crossed homozygotic floxed mTOR mice (40) with mice expressing Cre recombinase fused with the estrogen receptor driven by the Nestin promoter (41). This line enabled the nuclear translocation of Cre recombinase upon tamoxifen administration.

We intraperitoneally administered tamoxifen to Nestin-CreERT2; mTOR f/+ and Nestin-CreERT2; mTOR f/f mice to induce recombination at 2 weeks of age, and the retina was subjected to analysis at 4 weeks post administration when the mice reached 6 weeks of age. The partial attenuation of the mTOR gene was validated by immunofluorescence (Fig. 6A-B'; filled and open arrowheads for presence and absence, respectively), and accordingly, the activation of the target substrate pS6 was lost as well (Fig. 6C-D'). Moreover, evident upregulation of GFAP expression was found in mTOR conditional knockout mice (Fig. 6E-F'), suggesting that the loss of mTOR induces neuroinflammation. An increased number of apoptotic cells was found in Nestin-CreERT2; mTORf/f mice (Fig. 6G-H'), further demonstrating that the signals mediated by mTOR are necessary for retinal cell survival.

Together, the data suggest that mTOR signalling is required for the maintenance of retinal functions and are consistent with the notion that IGF signalling plays an important role in cell survival.

Retinal degeneration progresses through intercellular communication

In this study, we conducted single-cell gene expression profiling in the Prom1-/- retina exposed to a short light stimulation and identified the earliest responsive cells and genes. We identified Igf1 as one of the key factors whose expression was altered by light stimulation. Combinatorial treatment with an endothelin receptor blocker and sustained IGF1 expression improves retinal survival and function during the progression of RP.

RP is one of the retinal dystrophies whose symptoms are characterised by the degeneration of RPE and/or photoreceptor cells. The causative RP genes are, in most cases, expressed in either or both types of cells (42). Accordingly, as the photoreceptor and RPE cells collaboratively regulate the redox state of the retinoid (2), complications with either the photoreceptors or the RPE cells eventually result in similar phenotypes of visual dysfunctions. As Prom1 is mainly expressed in photoreceptor cells (Fig. 1E), it is reasonable to conclude that rod and cone photoreceptor cells are the cell types most severely affected by light stimulation, with changes in the expression of several genes (Table 1). However, in addition to photoreceptor cells, our scRNA-seq analysis identified astrocytes and Müller glial cells as the cell types where a number of genes were altered (Table 1). These cells were assumably affected secondarily by the complications occurring in the photoreceptor cells, considering that Prom1 is expressed in photoreceptor cells (Fig. 1E). The present study successfully identified the genes and cells affected at the onset of retinal degeneration. Importantly, we demonstrate that photoreceptor degeneration does not occur cell-autonomously but through cell‒cell communication with the surrounding cells.

Such intercellular communication and the activation of glial cells have been observed in other RP mutants (17, 43), and one of the strong candidates involved in this interaction is ET-2, produced by the Edn2 gene. Edn2/ET-2 is a common inflammatory factor that activates glial cells in the central nervous system (44), and light stimulation triggers Edn2 expression in rod photoreceptor cells (Fig. 2B) (6, 17). In glial cells, the responsive genes Gfap, Serpina3n, and S100a8/a9, which are inflammation-related and/or stress-responsive, are induced (23, 24, 45) (Fig. 2B, C). These reactive glial cells, in turn, release neurotrophic factors to promote the survival of surrounding cells (46) and phagocytose dead photoreceptors (47), thereby playing a positive role in retinal homeostasis.

However, one serious paradox of excessive activation of glial cells is that they induce the glial scar, which is a cell aggregate that causes a spatial obstruction to the retina, and retinal function is rather perturbed (48). Moreover, the activation of ET-2 exerts profound effects on the development and homeostasis of the retinal vasculature, perturbing vascular development (22), inducing the constriction of retinal venules (6, 21), and injuring the blood‒retinal barrier (49). Targeting gliosis is therefore a potential clinical strategy to delay disease progression and ameliorate associated symptoms (50, 51), and ET-2 is a strong target for this purpose.

Upregulation of the endothelin pathway has been found in a number of other mutant retinas causing RP, including those of rd10 mice, where phosphodiesterase 6b (Pde6b) is mutated (17), endothelin signal antagonists are adequate to ameliorate the degenerating phenotype (17, 52), and diabetic model db/db mice carrying a mutation in the leptin receptor gene (53). Therefore, it is highly assumed that similar mechanisms are common during RP, and endothelin receptor antagonists are effective in delaying the symptoms.

This study additionally found several genes altered by light stimulation. For instance, Hddc3 and Zfp69, whose expression is downregulated under light conditions, are involved in ferroptosis (54) and insulin sensitivity during diabetes (55, 56), respectively (Fig. 2C). Notably, the downregulation of Zfp69 is also found in db/db mice (57), which suggests that metabolic disorder occurs at the onset of retinopathy in the Prom1-/- retina. In addition, Gm26782 and Gm21887, encoding long noncoding RNAs (lncRNAs), were changed in some of the retinal subtypes (Fig. 2C). The functions of these genes could be explored during photoreceptor degeneration.

However, unlike Edn2, Hddc3, Zfp69 and the lnRNA genes were not commonly found in scRNA-seq analyses with other gene mutants (58–60). These differences may reflect the diversity in the severity of the phenotypes in each gene mutation.

IGF1 plays a protective role in photoreceptor cells

IGF1 is a versatile signalling molecule involved in several biological events, including differentiation (61, 62), proliferation (63), cell motility (64), metabolism (65) and survival (28). In particular, IGF, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and fibroblast growth factors (FGFs) act as neurotrophic factors in the central nervous system (66, 67), including the injured retina (28, 67), and exhibit a rescue effect on retinal cells. For instance, IGF1/IGF1R signalling plays a role in retinal vascularisation and growth (68, 69). Conversely, attenuation of the Igf1 gene (27, 28, 70) leads to blockade of autophagy and causes neuroinflammation (27). Moreover, knockout of the Igf1r gene (29) causes vision loss by altering the metabolism of retinal, fatty acid, and phosphoinositide (29).

We found that Igf1 gene expression was downregulated in rod photoreceptor cells and astrocytes by light stimulation (Fig. 2D). At this stage, pS6 expression was not found in either the Prom1+/- or Prom1-/- photoreceptor cells, presumably because Igf1 expression was too low and differences in expression could not be assessed with pS6. In the 3-week-old retina, the localisation of pS6 at photoreceptor cells became evident in the Prom1+/- retina (Fig. 3B). However, in the Prom1-/- retina, light stimulation inhibited the localisation of pS6 in photoreceptor cells (Fig. 3E). Furthermore, the decrease in pS6 in photoreceptor cells was found to be specific to light-stimulated conditions (Fig. 3F). We referred to our previous transcriptome analysis of the whole retina and found that Igf1 expression was downregulated (6). Thus, the downregulation of the Igf1 gene is consistent at least in the Prom1-KO retina. One possible mechanism for this phenotype is that the properties of the cells producing IGF1, either rod photoreceptors or astrocytes, were altered by the inflammatory responses, making IGF1 production difficult. The administration of Igf1 in the retina by AAV (Fig. 4E) successfully restored retinal survival and function (Fig. 4I, 4I’, 5C, 5D).

Photoreceptor cells are where aerobic glycolysis is active (32), and IGF signalling has been shown to play an essential role in this metabolic process (29). Therefore, it is reasonable that the IGF signal was partially inactivated in degenerating photoreceptor cells, and the compensation of IGF1 reactivates glycolysis and affects the NADH/NAD⁺ ratio (Fig. 4O).

IGF evokes several intracellular downstream signals, including those mediated by PI3K/AKT, mTOR, and ERK (71). Our analysis revealed that mTOR-mediated signalling is, at least in part, essential for retinal survival (Fig. 6). mTOR signalling has been shown to generally play essential roles in cell survival (30), and our findings are in good agreement with this notion (Fig. 4I, I’). Moreover, mTOR has been shown to be essential for autophagy (72). Therefore, the involvement of mTOR in IGF-induced cell survival provides a reasonable explanation for retinal survival.

However, one caveat for IGF activity is that there are also reports demonstrating the negative effects of IGF1 on the retina, where sustained IGF1 expression by a transgene induces retinal cell death and gliosis (73). In addition, local upregulation of IGF1 has been shown to trigger blood‒retinal barrier breakdown, and accordingly, humans with retinopathy with marked gliosis exhibited upregulation of IGF1R expression (74). However, in these studies, the analysed stages were elderly, more than 3 months old in mice, and older than 80 years old in humans. Therefore, one possible interpretation for these seeming contradictory phenotypes is that while cells require IGF activity, the excess level of IGF1 expression causes intense glycolysis, leading to earlier exhaustion. Therefore, to ensure the positive aspect of IGF signal for the retina's survival, it is necessary to control the IGF intensity quantitatively, and the establishment of its administration method is awaited.

Our functional analysis suggested that the sustained expression of IGF1 by AAV combined with treatment with endothelin receptor blockers tends to improve retinal function (Fig. 5). Considering that IGF activates aerobic glucose metabolism to maintain photoreceptor survival in a cell-autonomous manner, whereas endothelin receptor antagonists block neuroinflammation and glial hyperactivation, these two signals function in a complementary, but not redundant, manner to ameliorate the degenerative phenotype of the retina.

In exploring therapeutic methods for RP, one of the prominent methods currently established is the transplantation of retinal pigmented epithelium cells differentiated from stem cells into the degenerated area (75). Gene transfer into patients, so-called gene therapy (76–78), is also in the clinical stage. While regenerative techniques and gene compensation are expected to be fundamental therapeutic methods, there is still a need for more generally applicable treatments that target the causative gene. The method we propose in this study meets these requirements, and targeting molecules acting in the extracellular space is particularly useful to ensure drug accessibility.

Animals and treatments

Generation of the Prom1 mutant mice, where the locus has been replaced with the LacZ gene, was described elsewhere (79), and the stock can be found at the following site (https://large.riken.jp/distribution/mutant-list.html). The conditional knockout line of mTOR (Mtor < tm1.2Koz>/J) was purchased from Jackson Laboratory. Cre-ERT2 driven by the Nestin promoter (Tg(Nes-cre/ERT2)5.1 Imayo) (41) was distributed from Riken BioResource Center (RBRC05999; https://mus.brc.riken.jp/ja/order) under approval from Itaru Imayoshi.

Mice were usually reared at 20°C with a 12 hour-dark/light cycle with ad libitum access to food and water. To examine the effect of light stimulation, the pupils were reared in persistent dark condition until the experiment, the eyelids of young mice whose eyes were still closed were incised with a scalpel, and the eyes were forcibly opened. Pupils were dilated with topical application of 0.5% tropicamide and 0.5% phenylephrine (Santen; Mydrin-P®) to efficiently introduce light stimulation into the retina. The mice were reared under LED light at 15,000 lux for 3 hours.

Single-cell RNA sequencing analysis

For scRNA sequencing analysis, 2 male pupils with the homozygotic Prom1 mutation born from the same mother mouse were obtained. One of them was subjected to light stimulation at 11 days old for 3 hours as stated in the previous section and was immediately returned to the cage where the siblings were reared to recover overnight. The mice were sacrificed the next day, and the extracted retinas were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation; #PDS). We successfully isolated approximately 2x10⁵ cells from one retina with a viability of 80%.

For the construction of the scRNA-seq library, the Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics) was used according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 at a read length of 28x90 to yield a minimum of 20,000 reads per cell for 10,000 cells. The resulting raw data were processed by Cell Ranger 6.0.0 (10x Genomics).

The gene reads were acquired from 10,311 and 12,091 cells under dark and light conditions, respectively. Among them, 596 (5.8%; dark) and 704 (5.8%; light) cells were eliminated from the analysis objects, as these cells had 1000 or less unique molecular identifiers (UMIs), with 256 or fewer genes detected, or with more than 10% of mitochondria-derived genes. Next, each cell was categorised based on the representative genes that are specifically expressed in each cell type (80, 81), with the order of RPE, endothelial cells, microglia, horizontal cells, ganglion cells, Müller cells, cone photoreceptors, bipolar cells, astrocytes, amacrine cells, and rod photoreceptor cells. The gene list showing the fold changes and p values of each gene is in Supplementary Table 1.

RNA extraction and expression analysis

For RT‒qPCR, RNA and cDNA were prepared by using NucleoSpin® RNA (MACHEREY‑NAGEL; 740955) and PrimeScript RT Master Mix (Takara, RR036), respectively. Quantitative PCR was performed on a CFX qPCR machine (Bio-Rad), and the primer sequences are listed in Supplementary Tab. S2. Amplification data were analysed using the comparative Ct method, and the expression of each gene was normalised to that of RhoA.

Tissue analysis

For immunofluorescence, the eyeball was extracted at the indicated stage and fixed with paraformaldehyde. In 30 minutes, the lens was enucleated to encourage the penetration of the fixative and incubated for 30 more minutes. The tissues were incubated with 15% sucrose overnight and embedded in OCT compound (Sakura). The sectional samples were prepared in a Tissue Polar cryostat (Sakura fintek, Japan) at a thickness of 10 to 12 µm. The samples were incubated with primary antibodies overnight at 4°C and subsequently with secondary antibodies for 2 hours at room temperature. Nuclei were visualised by 4’,6-diamidino-2-phenylindole (DAPI). The antibodies used in this study are listed in Supplementary Tab. S2.

β-galactosidase staining was performed with staining solution containing 1 mM MgCl₂, 5 mM K₄[Fe^II(CN)₆], and 5 mM K₃[Fe^III(CN)₆] in PBS supplemented with 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal).

For TUNEL staining, sectioned specimens were incubated for 2 minutes with 0.1 M citric acid (pH 6.0) and were incubated in the reaction buffer containing in (Merck; S7105) 1xTdT reaction buffer, 1 mM CoCl₂, Terminal Transferase recombinant (Roche; 03333566001), and the signals were detected with anti-Digoxin antibody.

The NADH/NAD⁺ ratio was assayed by using a CycLex NAD⁺/NADH Colorimetric Assay Kit (MBL; CY-1253V2) following the manufacturer’s instructions, and the 450 nm absorbance was measured by an iMark microplate reader (Bio-Rad).

Preparation and injection of AAV

To generate AAVs carrying Gfp (control) or Igf1, we prepared pAAV-EGFP (for control; distributed from Addgene (#32395)) (82) and pAAV-IGF1, which was generated by replacing Gfp with the Igf1 gene. These AAVs were generated by transfecting either of the pAAV vectors with pHelper and pRC2-mi342 packaging vectors into HEK293 cells, and the AAVs were isolated four days posttransfection by using the AAVpro® Purification Kit Midi (TaKaRa #6675). The titres of the AAV were determined by PCR and were stocked as 5x10⁹/µl in PBS.

Intravitreal/subretinal injections into mice

Mice were anaesthetised with three types of mixed anaesthetic agents by intraperitoneal injection. A mixed anaesthetic was prepared with 0.75 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol. In addition, the cornea was anaesthetised with 0.4% oxybuprocaine hydrochloride ophthalmic solution (Santen; Benoxil). 10 µg of bosentan monohydrate (Tokyo Chemical Industry; B5118; in DMSO), 1 µg of human IGF1 (PeproTech; AF-100-11; in PBS), or 10⁹ genome copies of AAV were prepared alone or in mixtures and were introduced as 1 µl into the subretinal space or into the vitreous body of both eyes of each animal with a 35 G needle (Saito Medical Instruments Inc.). After AAV treatment, 0.5% levofloxacin ophthalmic solution (Santen; Cravit) was applied to the ocular surface to prevent infection, and 0.75 mg/kg atipamezole was intraperitoneally injected to reverse anaesthesia.

Functional analysis of the retina by ERG

ERG was performed by using the Neuropack S3 (Nihon Kohden, Co. Ltd. ), along with the LED Visual Stimulator LS-100 (Mayo Corporation) and the Hemisphere Stimulator (Mayo Corporation). All manipulations were performed under dim red light. Mice were adapted to the dark environment for at least 1 hour and were anaesthetised as described above. Tested animals were kept on a heating mat throughout the procedure to maintain body temperature. Pupils were dilated with a topical application of 0.5% tropicamide and 0.5% phenylephrine. Gold ring electrodes were placed on the corneal surfaces, and needle electrodes were inserted as ground and reference electrodes on the nose and tail, respectively. Responses to a 3162 cd/m² white light flash (1 ms) were amplified, filtered, and recorded. The a-wave amplitudes were measured from the baseline to the trough of the a-wave, and the b-wave was measured from the trough of the a-wave to the peak of the b-wave.

Conditional knockout of the mTOR gene

Mice carrying the flox-mTOR gene (40) were purchased from Jackson Laboratory (IMSR_JAX:011009), and either heterozygotic or homozygotic mutants were intraperitoneally administered 1 mg of (Z)-4-hydroxytamoxifen (Abcam ab141943) at 2 weeks of age and were harvested at 6 weeks of age by perfusion fixation (83).

Image processing and statistics

Images were captured using LSM980 confocal microscopes (Zeiss) and processed using Photoshop software (Adobe). Figures were prepared using Illustrator (Adobe) and Prism ver.10 (GraphPad). Numerical information in each graph is provided in Supplementary Table S3. Differences were evaluated using either a two-tailed Student’s t test (for comparisons between two groups) or one-way analysis of variance (ANOVA; for comparisons between more than two groups). Statistical comparisons with p < 0.05 were considered significant. p values (*p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) are indicated in each graph.

Funding

This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (21H02886 to Manabu Shirai; 23H02677 to Yasumasa Bessho; 23K14196 to Takuma Shinozuka; 20H03263 and 20H05036 to Noriaki Sasai), by R&D funding from the Japan Agency for Medical Research and Development (AMED) and Kyoto University, and by research grants from Novartis Pharma, the Suzuken Memorial Foundation and the Naito Foundation (to Noriaki Sasai).

Competing Interests

The authors declare that no competing interests exist.

Author Contributions

Noriaki Sasai conceived the study. Naoya Shigesada performed the majority of the experiments in this study with significant help from Naoya Shikada, Manabu Shirai, Fumiaki Higashijima, Kazuhiro Kimura and Yasumasa Bessho. Toru Kondo and Michinori Toriyama prepared the AAV-related materials and provided essential advice. Takuma Shinozuka conducted analyses on the mTOR-conditional mutants. Manabu Shirai, Yasumasa Bessho, Takuma Shinozuka and Noriaki Sasai acquired funding. Noriaki Sasai wrote the manuscript, and Naoya Shigesada and Takuma Shinozuka edited the manuscript. All authors agreed with the statements.

Data availability

The raw data for the single-cell RNA sequencing have been deposited at the DNA Data Bank of Japan (DDBJ) with the deposition number DRA016296.

Ethics approval

All animal experiments were performed under the approval of the animal ethics and welfare review panel of the Nara Institute of Science and Technology, and experiments were performed in accordance with the relevant internal and national regulations.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Acknowledgements

The authors thank the NGS core facility at the Research Institute for Microbial Diseases of Osaka University, particularly Daisuke Motooka, for their help with the single-cell RNA sequencing and data analysis, Chio Oka for comments on the manuscript, and the laboratory members for discussions and support.

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Table 1 is available in the Supplementary Files section

Tab.1.pdf
Tab. 1 The categorisationof the cells and changes in gene expression. The genes expressed in each cell type and the number of categorised cells.
Supplementarymaterialsfinal.pdf
Tab.S1.xlsx
Tab.S2.xlsx
Tab.S3.xlsx

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Combination of blockade of endothelin signalling and compensation of IGF1 expression confers protective effects on degenerating retina

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Introduction

Results

Single-cell expression profiling identified the cells and genes whose expression was altered by light exposure

Persistent IGF1 expression blocks photoreceptor cell death

mTOR-mediated signalling is required for photoreceptor survival

Discussion

Retinal degeneration progresses through intercellular communication

IGF1 plays a protective role in photoreceptor cells

Materials and Methods

Animals and treatments

Single-cell RNA sequencing analysis

RNA extraction and expression analysis

Tissue analysis

Preparation and injection of AAV

Intravitreal/subretinal injections into mice

Functional analysis of the retina by ERG

Image processing and statistics

Declarations

References

Table

Supplementary Files

Status:

Journal Publication

Version 1