STAT3 promotes survival of mutant photoreceptors in inherited photoreceptor degeneration models
Edited by Jeremy Nathans, Johns Hopkins University, Baltimore, MD, and approved November 26, 2014 (received for review June 16, 2014)
Significance
There is a great need for a therapy for inherited photoreceptor degenerations (IPDs) that would be effective irrespective of the affected gene. A treatment that slows photoreceptor (PR) death, even by 10–20%, could greatly extend the years of useful vision of patients with IPD for whom specific treatment is not available. Up-regulation of Stat3 has been reported in the retinas of many IPD models, but whether Stat3 expression is neuroprotective in mutant PRs has not been established. We show that Stat3 plays a strong prosurvival role in two distinct IPD models and that further augmentation of PR Stat3 expression slows PR death. These findings suggest the potential of Stat3 augmentation, for example, by recombinant adenoassociated virus (rAAV) vector-mediated gene therapy, as a treatment for IPDs.
Abstract
Inherited photoreceptor degenerations (IPDs), a group of incurable progressive blinding diseases, are caused by mutations in more than 200 genes, but little is known about the molecular pathogenesis of photoreceptor (PR) death. Increased retinal expression of STAT3 has been observed in response to many retinal insults, including IPDs, but the role of this increase in PR death is unknown. Here, we show that the expression of Stat3 is increased in PRs of the Tg(RHO P347S) and Prph2rds/+ mouse models of IPD and is activated by tyrosine phosphorylation. PR-specific deletion of Stat3 substantially accelerated PR degeneration in both mutant strains. In contrast, increased PR-specific expression of ROSA26 (R26) alleles encoding either WT STAT3 (Stat3wt) or the gain-of-function variant STAT3C (Stat3C) improved PR survival in both models. Moreover, PR signaling in Tg(RHO P347S) mice carrying either a R26-Stat3wt or R26-Stat3C allele demonstrated increased a-wave amplitude of the scotopic electroretinogram. Phosphorylation of STAT3 at tyrosine 705 was required for the prosurvival effect because an R26-Stat3Y705F allele was not protective. The prosurvival role of enhanced Stat3 activity was validated using recombinant adenoassociated virus (rAAV) vector-mediated PR Stat3 expression in Tg(RHO P347S) mice. Our findings (i) establish that the increase in endogenous PR Stat3 expression is a protective response in IPDs, (ii) suggest that therapeutic augmentation of PR Stat3 expression has potential as a common neuroprotective therapy for these disorders, and (iii) indicate that prosurvival molecules whose expression is increased in mutant PRs may have promise as novel therapies for IPDs.
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
Inherited photoreceptor degenerations (IPDs) are associated with striking genetic heterogeneity, resulting from mutations in more than 200 genes (RetNet Retinal Information Network, sph.uth.edu/retnet/). At least 20 different classes of proteins are affected, and a broad range of biological processes are disrupted (1, 2). Despite this diversity of genetic etiology, disease progression may be regulated by shared pathways that either promote or resist photoreceptor (PR) death (3). The identification of responses to a PR mutation that are common to many if not most IPDs include the increased expression of endothelin 2 (3, 4), accumulation of reactive oxygen species (ROS) (1, 5), increased expression of GFAP (3, 6, 7), and exponential death kinetics (8, 9). Some of these common events may have no influence on PR death, but others may represent general pathogenetic or survival responses that influence the rate of PR death, or even dictate it. The inhibition of common pathogenetic responses or the enhancement of shared prosurvival responses may constitute novel and potentially commonly applicable therapies for this largely untreatable group of inherited disorders.
To identify common prosurvival or pathogenetic responses in IPDs, we first examined changes in retinal gene expression in two well-characterized IPD models, Tg(RHO P347S) and Prph2rds/+ mice. We chose these two models because they are well-characterized, have substantially different rates of PR death, and disrupt proteins with widely different functions: the photopigment rhodopsin in Tg(RHO P347S) mice and the outer segment disk structural protein peripherin in Prph2rds/+ mice (10, 11). Given the diverse properties of proteins affected in these models, as well as their greatly differing rates of PR death, we reasoned that changes in gene expression shared by them may be common to other IPDs as well, irrespective of the biochemical nature of the defect or the rate of death.
We identified the up-regulation of genes in the IL-6 cytokine signaling pathway in retinas from both models. Activation of IL-6 signaling is initiated by binding of an IL-6 ligand to its specific receptor, forming a hexameric complex with the common coreceptor glycoprotein 130 (gp130), which, in turn, activates cytoplasmic janus kinases JAKs (JAK1, JAK2, JAK3, and Tyr kinase 2) (12). Activated JAK phosphorylates gp130 on its cytoplasmic domain, providing a docking site for the transcription factor STAT3, which is then phosphorylated by JAK on Tyr-705, leading to the formation of a phosphorylated STAT3Tyr705 (pSTAT3Tyr705) dimer that enters the nucleus to regulate target gene expression (12).
Numerous studies have suggested that activation of IL-6 signaling is important in the response to retinal insults. First, increased expression of Stat3 transcripts (3) or STAT3 and pSTAT3Tyr705 proteins (13–15) has been demonstrated in both Müller glia (15) and PRs (14, 15) of IPD and light-damaged retinas (3, 13–15). Second, the intravitreal or subretinal administration of an IL-6 cytokine, such as leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF), improves PR survival of both IPD and light-damaged retinas (16–22), and is accompanied by STAT3 phosphorylation in Müller cells (16, 17, 20, 22) and PRs (16, 17) of stressed retinas. Third, antagonism of the LIF receptor attenuates the protective effect on light damage of subtoxic light preconditioning (23). Fourth, PR death is more rapid in LIF-null mice subject to light damage (24) or carrying the VPP rhodopsin mutations (13). Fifth, the common coreceptor for IL-6 cytokine signaling, gp130, is required for the protective effects of LIF and CNTF in both light-damaged and IPD retinas: The deletion of gp130 significantly impairs the protective effect of subtoxic light preconditioning in PRs subjected to bright light (25), accelerates PR death in the VPP mouse (25), and attenuates the protective effect of lentivirus-mediated human CNTF expression from retinal pigment epithelium in Rds+/P216L mice (17). Sixth, elevated pSTAT3Tyr705 expression is observed following retinal detachment, possibly in response to IL-6 acting as a neuroprotectant. Subretinally injected anti–IL-6 neutralizing antibody accelerates PR death, whereas subretinally injected IL-6 prolongs PR survival (26).
Despite the circumstantial evidence suggesting that increased STAT3/pSTAT3Tyr705 expression has a neuroprotective role in IPDs, a mechanistic link to increased PR survival has not been demonstrated. IL-6 signaling downstream of the IL-6 receptor complex can also activate ERK1/2 and PI3K/AKT signaling (27), and these pathways alone could mediate the improved PR survival observed with LIF and CNTF administration. Therefore, to define the role of increased Stat3 signaling in the death of mutant PRs, we used the Cre/LoxP system to generate mice specifically lacking Stat3 function in the mutant PRs of both Tg(RHO P347S) and Prph2rds/+ retinas. We also augmented the expression of PR Stat3 in both mutants. Our findings demonstrate that increased Stat3 expression in mutant PRs is neuroprotective in these two biochemically distinct IPDs, suggesting the potential of PR Stat3 augmentation as a therapy for IPDs, irrespective of the causative gene.
Results
Stat3 Expression Is Elevated in Retinas from Tg(RHO P347S) and Prph2rds/+ Mice.
To identify differentially expressed genes in IPDs, we analyzed gene expression in retinas from two IPD models, Tg(RHO P347S) and Prph2rds/+ mice, using microarrays and quantitative PCR (qPCR). To minimize changes due to gross disruption of retinal structure and function, we examined both models at an early stage of degeneration: Tg(RHO P347S) mice at postnatal day 21 (PN21) and Prph2rds/+ mice at 7 wk, when 70% and 90%, respectively, of the mutant PRs were still alive. We found that the expression of nine IL-6 signaling pathway genes was up-regulated in retinas from both models (Table S1), suggesting that this pathway may play an important role in IPDs.
By immunoblotting of total retinal extracts, we found the expression of both STAT3 and pSTAT3Tyr705 to be increased in both IPD models vs. age-matched WT controls: STAT3 and pSTAT3Tyr705 expression was 2.6-fold (n = 3, P < 0.001) and 1.6-fold (n = 3, P < 0.05) greater in Tg(RHO P347S) retinas, respectively, at PN21, and it was 2.6-fold (n = 3, P < 0.05) and 1.7-fold (n = 3, P < 0.05) greater in Prph2rds/+ retinas, respectively, at 7 wk (Fig. 1A).
Fig. 1.
Stat3 Expression Is Increased in Both Müller Glia and Mutant PRs in Tg(RHO P347S) and Prph2rds/+ Mice.
To define retinal cell types expressing the increased levels of STAT3 and pSTAT3Tyr705, we performed immunofluorescence (IF) staining on Tg(RHO P347S) retinas at PN21, on Prph2rds/+ retinas at 7 wk of age, and on age-matched WT controls. Increased STAT3 expression was observed in Müller glia in both IPD models (Fig. 1B), as reported in other IPDs (15). Notable foci of substantially increased pSTAT3Tyr705 staining were seen in some Müller glia and in the contiguous PR layer in both mutant retinas (Fig. 1B). In contrast, it was less clear whether increases in STAT3 and pSTAT3Tyr705 staining were present in the PR layer (Fig. 1B).
To determine more precisely whether the expression of Stat3 was increased in mutant PRs, and to quantify its expression in both the outer nuclear layer (ONL) and inner nuclear layer (INL), we used laser-capture microdissection to isolate these layers from the retinas of PN21 Tg(RHO P347S) mice, 7-wk-old Prph2rds/+ mice, and age-matched WT control mice. Total mRNA was isolated for qPCR analysis, and the layer-specific biomarkers Chx10 and Rho were used to confirm specific capture of each layer (Fig. 1C). We observed significant increases in the abundance of Stat3 mRNA in the ONL of both Tg(RHO P347S) mice (5.3-fold, n = 3; P < 0.001) and Prph2rds/+ mice (2.4-fold, n = 3; P < 0.05) vs. age-matched WT controls. We also found the expression of Stat3 mRNA to be increased in the INL of both mutants: 1.8-fold (n = 3; P < 0.01) and 1.5-fold (n = 3; P < 0.05) in Tg(RHO P347S) and Prph2rds/+ retinas, respectively. In summary, the expression of Stat3 was increased in both mutant PRs and Müller glia of both IPD models.
PR-Specific Deletion of Stat3 Decreases PR Survival in Both Tg(RHO P347S) and Prph2rds/+ Mice.
To establish whether the increase in Stat3 expression in mutant PRs is a pathogenetic or prosurvival response, we deleted Stat3 from the PRs of both Tg(RHO P347S) and Prph2rds/+ mice. To this end, we first introduced homozygous Stat3fl/fl alleles (28) into both Tg(RHO P347S) and Prph2rds/+ backgrounds and then crossed them to mice with PR-specific expression of Cre [Tg(Opsin-iCre)] (29). Cre protein expression was detectable in the PR layer by PN10, and was observed throughout the PR layer by PN15, when the majority of mutant PRs are still alive in both models (10, 11) (Fig. S1). Cre expression did not appear to be detrimental to PRs, because normal retinal and PR morphology and normal ONL thickness were observed in Tg(Opsin-iCre) mice at both PN40 and 6 mo (Fig. 2 A and G), in agreement with other reports (29, 30).
Fig. 2.
We found that STAT3 expression in whole retinal extracts was significantly reduced following Cre-mediated deletion of the PR Stat3 gene (Fig. S2). Moreover, IF staining demonstrated that STAT3 was virtually absent from the PRs of Stat3fl/fl; Tg(RHO P347S); Tg(Opsin-iCre) retinas at PN20 and from the PRs of Stat3fl/fl; Prph2rds/+; Tg(Opsin-iCre) retinas at 6 mo (Fig. S3). Importantly, no significant differences in gross retinal or PR morphology, or in ONL thickness, were observed between Stat3fl/fl and Stat3fl/fl; Tg(Opsin-iCre) retinas at PN40 (n = 6; P = 0.86) or 6 mo (n = 4; P = 0.36) (Fig. 2 B and H), indicating that Stat3 is not required for the survival of mature PRs. Furthermore, PR-specific expression of Cre did not influence PR degeneration in either model because there was no difference in the degree of PR death between Tg(RHO P347S); Tg(Opsin-iCre) and Tg(RHO P347S) mice at PN30 (n = 7; P = 0.82) or between Prph2rds/+; Tg(Opsin-iCre) and Prph2rds/+ mice at 6 mo (n = 13; P = 0.27) (Fig. 2 C and I).
In contrast to the situation on a WT background, we found that the deletion of Stat3 from mutant PRs substantially increased PR death in both IPD models (Fig. 2 D–F and J–L). ONL thickness decreased by 40% and 67% in Stat3fl/fl; Tg(RHO P347S); Tg(Opsin-iCre) vs. Stat3fl/fl; Tg(RHO P347S) retinas at PN20 (n = 6; P < 0.01) and PN30 (n = 5; P < 0.001), respectively, and by 17% and 50% in Stat3fl/fl; Prph2rds/+; Tg(Opsin-iCre) vs. Stat3fl/fl; Prph2rds/+ retinas at 2 mo (n = 7; P < 0.01) and 6 mo (n = 9; P < 0.001), respectively. These findings establish that the increased endogenous expression of PR Stat3 is a major prosurvival response in mutant PRs.
Augmentation of PR Stat3 Expression from Rosa26-Stat3 Alleles Increases PR Survival.
Because the loss of Stat3 function was detrimental to the survival of mutant PRs, we next asked whether additional PR Stat3 expression, beyond that conferred by endogenous Stat3 genes, would enhance mutant PR survival. To this end, we used the Cre-conditional ROSA26 (R26) knock-in system (31, 32) to express either a third WT (Stat3wt), a gain-of-function (Stat3C) (33), or a dominant negative (Stat3Y705F) allele, specifically in PRs (Fig. S4). The Cys residues in STAT3C (34) stabilize formation of the active tyrosine-phosphorylated homodimeric complex on DNA by reducing efficiency of dephosphorylation at Tyr-705 and by increasing DNA-binding affinity of the dimer (35, 36). In contrast, STAT3Y705F cannot be phosphorylated at Tyr-705 and interferes with phosphorylation and dimerization of WT STAT3, therefore acting in a dominant negative fashion (37). The three R26-Stat3 alleles were strongly expressed, as confirmed by RT-PCR (Fig. S5). The abundance of total retinal Stat3 mRNA increased by 39% in R26+/LSL-Stat3wt; Tg(Opsin-iCre) mice (n = 3; P < 0.01), by 52% in R26+/LSL-Stat3C; Tg(Opsin-iCre) mice (n = 3; P < 0.01), and by 42% in R26+/LSL-Stat3Y705F; Tg(Opsin-iCre) mice (n = 3; P < 0.01), compared with their age-matched Tg(Opsin-iCre)-negative littermates (Fig. S6).
To determine whether expression of the three R26-Stat3 alleles improved PR survival in IPDs, we expressed them in Tg(RHO P347S); Tg(Opsin-iCre) and Prph2rds/+; Tg(Opsin-iCre) retinas. We first demonstrated that expression of all three alleles had no effect on the morphology or viability of the PRs of Tg(Opsin-iCre) mice, at least up to 6 mo of age (Fig. 3 A, E, F, and J and Fig. S7). However, expression of the Stat3wt allele led to a highly significant 26% (n = 14; P < 0.001) increase of PR survival in R26+/LSL-Stat3wt; Tg(RHO P347S); Tg(Opsin-iCre) vs. R26+/LSL-Stat3wt; Tg(RHO P347S) retinas at PN30 (Fig. 3 B and E). Increased survival was even greater with Stat3C, where ONL thickness increased by 39% (n = 12; P < 0.001) in R26+/LSL-Stat3C; Tg(RHO P347S); Tg(Opsin-iCre) vs. R26+/LSL-Stat3C; Tg(RHO P347S) retinas at PN30 (Fig. 3 C and E). In contrast, the Stat3Y705F allele had no effect on ONL thickness in R26+/LSL-Stat3Y705F; Tg(RHO P347S); Tg(Opsin-iCre) vs. R26+/LSL-Stat3Y705F; Tg(RHO P347S) mice at PN30 (n = 13; P = 0.61) (Fig. 3 D and E), suggesting that tyrosine phosphorylation and dimerization of STAT3 are required for its prosurvival effect. Comparable effects were observed in Prph2rds/+ mice. At 6 mo, ONL thickness in Prph2rds/+ retinas was increased by 14% (n = 10; P < 0.05) with expression of the Stat3wt allele (Fig. 3 G and J) and by 19% (n = 7; P < 0.001) with expression of the Stat3C allele (Fig. 3 H and J), whereas expression of the Stat3Y705F allele lacked a prosurvival effect (n = 5; P = 0.53) (Fig. 3 I and J). These findings suggest that the protective effect of augmented Stat3 expression on mutant PRs may be relevant to IPDs that result from a broad range of IPD genes.
Fig. 3.
We further investigated whether the increased PR survival was associated with enhanced retinal electrophysiological responses in Tg(RHO P347S) mice expressing WT or mutationally activated Stat3. Specifically, we compared the electroretinograms (ERGs) of R26+/LSL-Stat3wt; Tg(RHO P347S); Tg(Opsin-iCre) and R26+/LSL-Stat3C; Tg(RHO P347S); Tg(Opsin-iCre) mice with their corresponding Tg(Opsin-iCre)-negative littermates. We observed a significant increase of the a-wave amplitude in scotopic ERG recordings in Tg(RHO P347S) mice expressing either the Stat3wt allele at PN30 (n = 10; P < 0.05) or the Stat3C allele at both PN20 (n = 16; P < 0.05) and PN30 (n = 15; P < 0.001) (Fig. 4). By PN45, however, when the ONL was reduced to two to three rows of nuclei, the effect of the increased expression of either R26-Stat3 allele was no longer apparent (Fig. 4).
Fig. 4.
PR Expression of the rAAV8(Y733F)-hGRK1-Stat3-FLAG Vector Increases PR Survival and Retinal Electrophysiological Responses in Tg(RHO P347S) Mice.
To validate the protective effect of enhanced PR-specific Stat3 expression in IPDs, we also augmented Stat3 expression in Tg(RHO P347S) PRs using a recombinant adenoassociated virus (rAAV) vector. We first examined the effect on the retina of subretinal injection of an rAAV control vector, rAAV8(Y733F)-hGRK1-EGFP. This vector was injected into one eye of WT or Tg(RHO P347S) mice, and the effect was compared with the contralateral uninjected eye. We observed no differences in retinal morphology, ONL thickness, or scotopic a-wave amplitude in the injected vs. contralateral eye in either WT or Tg(RHO P347S) mice (Fig. S8). These results demonstrate that injection of an rAAV8(Y733F) vector does not, per se, have any apparent effect on Tg(RHO P347S) PRs, and that the injection wound itself does not enhance PR survival. These findings also support the use of an uninjected eye as an appropriate control, as has been demonstrated by others (38–40).
To determine whether rAAV-mediated enhancement of Stat3 expression in Tg(RHO P347S) PRs improves their survival, we subretinally injected an rAAV8(Y733F)-hGRK1-Stat3-FLAG vector in one eye of mutant and WT mice at PN14. We first established that rAAV vector-mediated STAT3 expression, driven by the human G-protein receptor kinase 1 promoter, was specific to PR cell bodies and inner segments (Fig. 5A). We then demonstrated that the injection of rAAV8(Y733F)-hGRK1-Stat3-FLAG vector had no detrimental effect on WT PRs. WT retinas injected with this rAAV vector had normal retinal and PR morphology, ONL thickness, and a-waves responses in scotopic ERG recordings at PN60, comparing injected and uninjected eyes (Fig. S9).
Fig. 5.
In confirmation of the prosurvival effect of augmenting PR Stat3 expression using R26-Stat3 alleles, we observed a mean 15 ± 5% increase (n = 18; P < 0.01) in ONL thickness at PN60 in rAAV-injected Tg(RHO P347S) eyes vs. uninjected eyes (Fig. 5 B–D). In addition, the physiological responses of the mutant PRs were improved: at PN60, the scotopic a-wave amplitude under low light intensity was significantly increased in the injected eye (n = 18; P < 0.001) compared with the uninjected control eye (Fig. 5E). Smaller improvements in a-wave amplitude were observed at higher light intensities, suggesting that the PRs with improved survival are mostly rods. Thus, rAAV vector-mediated enhancement of PR Stat3 expression has a significant protective effect on the mutant PRs of Tg(RHO P347S) mice.
Discussion
Our findings firmly establish that increased expression of PR STAT3 and pSTAT3Tyr705 has a prosurvival role in mutant PRs. First, we found that endogenous Stat3 expression was significantly increased in the mutant PRs from two unrelated IPD models, Tg(RHO P347S) and Prph2rds/+ mice. Second, deletion of Stat3 in mutant PRs greatly increased the degree of PR death. Third, augmentation of either STAT3wt or STAT3C expression, using R26 transgenes, significantly enhanced mutant PR survival in both Tg(RHO P347S) and Prph2rds/+ mice and improved retinal electrophysiology in the one model tested [Tg(RHO P347S)]. Taken together, these results suggest a cell-autonomous protective role for STAT3 in mutant PRs. The role of the Müller cell Stat3 activation in IPDs is unknown. Our findings, together with other work suggesting that increased pSTAT3Tyr705 expression may be protective against a variety of neuronal insults (41–43), indicate that enhanced Stat3 signaling merits study as a general neuroprotectant.
The mechanism underlying the protective effect of STAT3 in IPDs remains to be defined. The fact that Tyr705 phosphorylation is required for this effect suggests that pSTAT3Tyr705 target genes with gamma interferon activation site (GAS) elements (TTN5AA) in their promoters are strong candidates as mediators of the protection. Identification of these target genes may lead to molecular therapies with selective and strong prosurvival effects. Known pSTAT3Tyr705 downstream genes that may be responsible for the Stat3-dependent effect on mutant PR survival include TP53, Mcl-1, c-Fos, and Bcl-2 (44, 45), all of which have GAS sites in their promoter. pSTAT3Tyr705 may act as a transcriptional repressor of p53 expression in mutant PRs, because STAT3 inhibition in cancer cells leads to up-regulation of p53 expression and p53-mediated apoptosis (46). Mcl-1, a pSTAT3Tyr705 target in cancer cells, has also been suggested to play a protective role in the nervous system. Mcl-1 deletion sensitizes cultured postmitotic neurons to acute DNA damage (47), and the ubiquitination and degradation of MCL-1 proteins trigger apoptosis of cerebellar granule neurons (48). c-Fos suppresses c-Jun–mediated neuronal apoptosis by reducing the expression of c-Jun/ATF2 target genes (49), and Bcl-2 overexpression in mutant PRs transiently delays PR death (50, 51).
The protective effect of pSTAT3Tyr705 may also be mediated by an increase in the abundance of unphosphorylated STAT3 (U-STAT3): Stat3 expression is activated by pSTAT3Tyr705 dimers, and sustained activation may lead to U-STAT3 accumulation (52) in mutant PRs. U-STAT3 can bind unphosphorylated NF-κB, thereby facilitating activation of a subset of NF-κB target genes, including the neuroprotective cytokine gene Ccl5 (53, 54). STAT3 monophosphorylated at serine 727, pStat3Ser727, may also play a significant role in IPD pathogenesis. pStat3Ser727 is found in mitochondria, where it regulates the activity of complexes I and II of the electron transport chain, and the production of ROS (55, 56). Indeed, increased ROS generation is a common response to PR mutations (1, 5).
Our demonstration of a role for PR expression of Stat3 in mutant PR survival suggests that genetic and pharmacological strategies to augment the elevated STAT3 activity further merit examination in other IPD models. The improved PR survival and retinal electrophysiological responses in Tg(RHO P347S) mice injected with rAAV-Stat3 vectors highlight the potential of viral-mediated PR Stat3 delivery as a novel therapeutic approach for IPDs. Although we observed no adverse effects of increased Stat3 expression on PR or retinal morphology for up to 6 mo, longer periods of posttreatment surveillance and vision testing are required.
Our identification of enhanced Stat3 expression as neuroprotective in mutant PRs suggests that other common IPD response genes may play a similar role. For example, the expression of both endothelin-2 and FGF2 is strongly increased in the retina of many IPD and light-damaged models (3, 4, 7, 13), and both have been shown to increase mutant PR survival (4, 57, 58). Moreover, FGF-2 expression in light-damaged and IPD retinas is regulated by LIF (13, 24), and pSTAT3Tyr705 binds directly to the FGF2 promoter (59), further suggesting that as a putative pSTAT3Tyr705 downstream gene, FGF2 has great potential to be a future therapeutic target in IPDs. Finally, the expression of several isoforms of metallothionein (MT) is increased in IPD and light-damaged retinas (3, 7, 60, 61), and the deletion of MT-3 increases PR death in light damage (62).
In conclusion, our findings suggest that augmentation of the expression of Stat3 or one or more of its downstream target genes in mutant PRs has potential as a general therapy for IPDs. Even if a specific and effective treatment, such as gene replacement therapy, is available, slowing the progression of PR death may have an important impact on the maintenance of vision while a precise genetic diagnosis is being made and access to gene therapy is obtained.
Methods
Mice.
Prph2rds/rds (C3A.BLiA-Pde6b+.O20-Prph2Rd2/J) mice, their corresponding control stain Prph2+/+ (C3A.BLiA-Pde6b+/J), and C57BL/6J mice were obtained from the Jackson Laboratory. Prph2rds/+ mice were generated by crossing Prph2rds/rds and Prph2+/+ mice. Tg(RHO P347S) (C57BL/6J) mice were provided by Tiansen Li, Harvard University, Boston, MA, Stat3flox/flox (BALBC/A) mice were provided by Christine Watson, University of Cambridge, Cambridge, UK., Tg(Opsin-iCre) (C57BL/6.SJL-Pde6b+) mice were provided by Ching-Kang Chen (Virginia Commonwealth University, Richmond, VA), and R26+/LSL-Stat3C (C57BL/6J.BALB/c) mice were provided by Klaus Rajewsky, Harvard University, Boston, MA. Animals were maintained with 12-h light/dark cycles and treated in accordance with guidelines and principles outlined by the Animal Care Committee at The Hospital for Sick Children (Toronto).
Immunoblotting.
Whole retinal protein samples were extracted from freshly dissected retinas. One hundred micrograms of total protein was loaded in each lane of 10% (wt/vol) SDS/PAGE gels and transferred to Hybond-C extra membrane (Amersham). Membranes were incubated with the following antibodies at 4 °C overnight: anti-STAT3 at a 1:2,000 dilution (Cell Signaling), anti-pSTAT3Tyr705 at a 1:2,000 dilution (Cell Signaling), anti–β-actin at a 1:5,000 dilution (Abcam), anti-Cre at a 1:10,000 dilution (Novagen), and anti-CHX10 at a 1:5,000 dilution (homemade). A secondary antibody to rabbit IgG with HRP conjugates (1:5,000; Sigma–Aldrich) was used, followed by detection using an ECL system.
Immunostaining.
Paraffin sections from WT, Prph2rds/+, and Tg(RHO P347S) eyes were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling for 20 min in 10 mM sodium citrate, pH 6.0, solution. They were then incubated overnight at 4 °C with the indicated primary antibodies: anti-STAT3 at a 1:100 dilution (Cell Signaling), anti-pSTAT3Tyr705 at a 1:100 dilution (Cell Signaling), anti-p27kip1 at a 1:500 dilution (BD Biosciences), anti-Cre at a 1:400 dilution (Novagen), and anti-FLAG at a 1:100 dilution (Santa Cruz Biotechnology). Appropriate secondary antibodies, conjugated to chromofluor, were then applied: For STAT3, pSTAT3Tyr705, Cre, and FLAG in a 1:100 dilution of goat anti-rabbit Cy3 (Invitrogen) was used, whereas for p27kip1, a 1:100 dilution of goat anti-mouse Alexa488 (Invitrogen) was used. Sections were then washed in PBS and mounted in immunomount (Shandon-Mount; Thermo Scientific). Stained sections were visualized using a Zeiss LSM510 Laser Scanning Confocal Microscope, and images were acquired using the LSM510 software package (Zeiss).
Laser-Capture Microdissection.
Enucleated eyes were fresh-frozen in optimal cutting temperature compound (Tissue-Tek; Miles), and 14-μm sections were cut using a Leica cryostat and placed on laser capture slides (Molecular Machines and Industries). Sections were stained using H&E to visualize retinal cell layers. The ONL and INL were isolated separately, according to the manufacturer’s instructions, using a Zeiss Axiovert 200 inverted fluorescence microscope equipped with an MMI Cellcut Laser Microdissection workstation and a Sony 3 chip CCD camera.
Retinal ONL Measurements.
Enucleated eyes were marked with a flamed needle to identify the superior (dorsal) hemisphere and were fixed in 2% (wt/vol) glutaraldehyde and 0.1 M phosphate buffer. The superior hemisphere was cut into three equivalent parts, and the rostral slices were embedded in Jembed 812 (Canemco). Sections (800 nm) were cut, stained with 1% toluidine blue, and imaged with a Leica DM1000 microscope and digital camera. The width of the ONL was measured using SigmaScan Pro (Systat Software, Inc.).
Electroretinography.
Mouse eyes were dilated by topical application of 0.25% hyoscine and dark-adapted overnight. Dilation was reinforced 30 min before testing. Mice were anesthetized by i.p. injection of ketamine/xylazine, based on their weight, and placed on a heating pad. A monopolar contact loop was placed on the surface of the cornea as the active electrode; needle electrodes under the scalp and in the tail served as a reference and ground, respectively. Full-field ERGs were recorded from both eyes following the International Society for Clinical Electrophysiology of Vision standard protocol adapted for mice. PR responses (a-waves) were elicited with flashes (0.02–200 cd⋅s⋅m−2), and signals were amplified and averaged on a personal computer-based recording system. Intermixed experimental and control littermates were measured on the same day, and the operator was blinded to the genotype of animals.
Subretinal Injection.
Mouse eyes were dilated by topical application of 1% atrophine sulfate ophthalmic solution (Bausch & Lomb) and 2.5% (wt/vol) phenylephrine hydrochloride ophthalmic solution (Akorn). Mice were anesthetized by i.p. injection with ketamine/xylazine, based on their weight, and placed on a heating pad. Eyes were punctured with a 30-gauge needle tip between the corneoscleral junction and the ora serrata into the vitreous cavity. Viral substance was injected into the subretinal space using a 33-gauge, 1.5-inch blunt needle attached to a 10-μL Hamilton syringe (Hamilton Company) under a dissecting microscope. A volume of 1 μL of undiluted rAAV8(Y733F)-hGRK1-EGFP or rAAV8(Y733F)-hGRK1-Stat3-FLAG viral particles (1 × 1012 vector genomes per milliliter) was injected in each eye, creating a retinal detachment or bleb. All mice were topically treated with tropicamide ointment (Alcon) following injection.
Statistical Analysis.
When two individual experimental groups were analyzed, statistical analyses were performed using unpaired two-tailed Student t tests. We used paired two-tailed Student t tests for analyses comparing rAAV8(Y733F)-hGRK1-EGFP or rAAV8(Y733F)-hGRK1-Stat3-FLAG injected retinas with the retinas of uninjected controls. Statistical significance was considered at P ≤ 0.05.
Acknowledgments
We thank Dr. T. Li (Harvard University) for providing Tg(RHO P347S) mice, Dr. C. J. Watson (University of Cambridge) for providing Stat3fl/fl mice, Dr. C. K. Chen (Virginia Commonwealth University) for providing Tg(Opsin-iCre) mice, and Dr. K. Rajewsky (Harvard University) for providing R26+/LSL-Stat3C mice. We also thank the Montreal Genome Centre for performing microarray analyses and the Toronto Center for Phenogenomics for generating R26+/LSL-Stat3wt and R26+/LSL-Stat3Y705F mice. This work was supported by an award from the Vision Science Research Program provided by the Ontario Student Opportunity Trust Fund (to K.J.) and by Grants MOP-7315 and IOP-54037 (to R.R.M.) from the Canadian Institutes of Health Research, the Canadian Genetic Diseases Network, and the Macula Vision Research Foundation. R.R.M. is a Tier 1 Canada Research Chair in Neurogenetics. The S.E.E. laboratory was supported by The Terry Fox Foundation and the Canadian Breast Cancer Foundation. The rAAV8(Y733F)-hGRK1-Stat3-FLAG virus was generated by the W.W.H. laboratory. W.W.H. acknowledges partial support of this work from NIH Grants P30EY021721 and R01EY17549 and grants from the Macular Vision Research Foundation; Overstreet Fund; and Research to Prevent Blindness, Inc. The subretinal injection and ERGs were performed by the Q.L. laboratory, supported, in part, by NIH Grant EY021752; the Overstreet Fund; and Research to Prevent Blindness, Inc.
Supporting Information
Supporting Information (PDF)
Supporting Information
- Download
- 1.05 MB
References
1
AN Bramall, AF Wright, SG Jacobson, RR McInnes, The genomic, biochemical, and cellular responses of the retina in inherited photoreceptor degenerations and prospects for the treatment of these disorders. Annu Rev Neurosci 33, 441–472 (2010).
2
AF Wright, CF Chakarova, MM Abd El-Aziz, SS Bhattacharya, Photoreceptor degeneration: Genetic and mechanistic dissection of a complex trait. Nat Rev Genet 11, 273–284 (2010).
3
A Rattner, J Nathans, The genomic response to retinal disease and injury: Evidence for endothelin signaling from photoreceptors to glia. J Neurosci 25, 4540–4549 (2005).
4
AN Bramall, et al., Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration. PLoS ONE 8, e58023 (2013).
5
F Doonan, G Groeger, TG Cotter, Preventing retinal apoptosis—Is there a common therapeutic theme? Exp Cell Res 318, 1278–1284 (2012).
6
AS Hackam, et al., Identification of gene expression changes associated with the progression of retinal degeneration in the rd1 mouse. Invest Ophthalmol Vis Sci 45, 2929–2942 (2004).
7
M Samardzija, et al., Activation of survival pathways in the degenerating retina of rd10 mice. Exp Eye Res 99, 17–26 (2012).
8
G Clarke, et al., A one-hit model of cell death in inherited neuronal degenerations. Nature 406, 195–199 (2000).
9
LR Pacione, MJ Szego, S Ikeda, PM Nishina, RR McInnes, Progress toward understanding the genetic and biochemical mechanisms of inherited photoreceptor degenerations. Annu Rev Neurosci 26, 657–700 (2003).
10
T Li, WK Snyder, JE Olsson, TP Dryja, Transgenic mice carrying the dominant rhodopsin mutation P347S: Evidence for defective vectorial transport of rhodopsin to the outer segments. Proc Natl Acad Sci USA 93, 14176–14181 (1996).
11
S Sanyal, A De Ruiter, RK Hawkins, Development and degeneration of retina in rds mutant mice: Light microscopy. J Comp Neurol 194, 193–207 (1980).
12
D Kamimura, K Ishihara, T Hirano, IL-6 signal transduction and its physiological roles: The signal orchestration model. Rev Physiol Biochem Pharmacol 149, 1–38 (2003).
13
S Joly, C Lange, M Thiersch, M Samardzija, C Grimm, Leukemia inhibitory factor extends the lifespan of injured photoreceptors in vivo. J Neurosci 28, 13765–13774 (2008).
14
M Samardzija, et al., Differential role of Jak-STAT signaling in retinal degenerations. FASEB J 20, 2411–2413 (2006).
15
K Schaeferhoff, et al., Induction of STAT3-related genes in fast degenerating cone photoreceptors of cpfl1 mice. Cell Mol Life Sci 67, 3173–3186 (2010).
16
Y Ueki, J Wang, S Chollangi, JD Ash, STAT3 activation in photoreceptors by leukemia inhibitory factor is associated with protection from light damage. J Neurochem 105, 784–796 (2008).
17
KD Rhee, et al., CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in Müller glial cells. Proc Natl Acad Sci USA 110, E4520–E4529 (2013).
18
D Bok, et al., Effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a P216L rds/peripherin mutation. Exp Eye Res 74, 719–735 (2002).
19
SP Huang, PK Lin, JH Liu, CN Khor, YJ Lee, Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats. J Biomed Sci 11, 37–48 (2004).
20
Y Song, et al., Photoreceptor protection by cardiotrophin-1 in transgenic rats with the rhodopsin mutation s334ter. Invest Ophthalmol Vis Sci 44, 4069–4075 (2003).
21
W Tao, et al., Encapsulated cell-based delivery of CNTF reduces photoreceptor degeneration in animal models of retinitis pigmentosa. Invest Ophthalmol Vis Sci 43, 3292–3298 (2002).
22
X Xia, et al., Oncostatin M protects rod and cone photoreceptors and promotes regeneration of cone outer segment in a rat model of retinal degeneration. PLoS ONE 6, e18282 (2011).
23
S Chollangi, J Wang, A Martin, J Quinn, JD Ash, Preconditioning-induced protection from oxidative injury is mediated by leukemia inhibitory factor receptor (LIFR) and its ligands in the retina. Neurobiol Dis 34, 535–544 (2009).
24
S Bürgi, M Samardzija, C Grimm, Endogenous leukemia inhibitory factor protects photoreceptor cells against light-induced degeneration. Mol Vis 15, 1631–1637 (2009).
25
Y Ueki, YZ Le, S Chollangi, W Muller, JD Ash, Preconditioning-induced protection of photoreceptors requires activation of the signal-transducing receptor gp130 in photoreceptors. Proc Natl Acad Sci USA 106, 21389–21394 (2009).
26
DY Chong, et al., Interleukin-6 as a photoreceptor neuroprotectant in an experimental model of retinal detachment. Invest Ophthalmol Vis Sci 49, 3193–3200 (2008).
27
R Eulenfeld, et al., Interleukin-6 signalling: More than Jaks and STATs. Eur J Cell Biol 91, 486–495 (2012).
28
T Alonzi, et al., Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver. Mol Cell Biol 21, 1621–1632 (2001).
29
S Li, et al., Rhodopsin-iCre transgenic mouse line for Cre-mediated rod-specific gene targeting. Genesis 41, 73–80 (2005).
30
P Barabas, et al., Role of ELOVL4 and very long-chain polyunsaturated fatty acids in mouse models of Stargardt type 3 retinal degeneration. Proc Natl Acad Sci USA 110, 5181–5186 (2013).
31
P Soriano, Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70–71 (1999).
32
S Srinivas, et al., Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4 (2001).
33
S Casola, et al., Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting. Proc Natl Acad Sci USA 103, 7396–7401 (2006).
34
JF Bromberg, et al., Stat3 as an oncogene. Cell 98, 295–303 (1999).
35
L Li, PE Shaw, Elevated activity of STAT3C due to higher DNA binding affinity of phosphotyrosine dimer rather than covalent dimer formation. J Biol Chem 281, 33172–33181 (2006).
36
FJ Liddle, JV Alvarez, V Poli, DA Frank, Tyrosine phosphorylation is required for functional activation of disulfide-containing constitutively active STAT mutants. Biochemistry 45, 5599–5605 (2006).
37
A Kaptein, V Paillard, M Saunders, Dominant negative stat3 mutant inhibits interleukin-6-induced Jak-STAT signal transduction. J Biol Chem 271, 5961–5964 (1996).
38
LL Molday, et al., RD3 gene delivery restores guanylate cyclase localization and rescues photoreceptors in the Rd3 mouse model of Leber congenital amaurosis 12. Hum Mol Genet 22, 3894–3905 (2013).
39
JJ Pang, et al., Long-term retinal function and structure rescue using capsid mutant AAV8 vector in the rd10 mouse, a model of recessive retinitis pigmentosa. Mol Ther 19, 234–242 (2011).
40
A Dinculescu, et al., Gene therapy for retinitis pigmentosa caused by MFRP mutations: Human phenotype and preliminary proof of concept. Hum Gene Ther 23, 367–376 (2012).
41
F Scheibe, O Klein, J Klose, J Priller, Mesenchymal stromal cells rescue cortical neurons from apoptotic cell death in an in vitro model of cerebral ischemia. Cell Mol Neurobiol 32, 567–576 (2012).
42
S Sharma, et al., IL-10 directly protects cortical neurons by activating PI-3 kinase and STAT-3 pathways. Brain Res 1373, 189–194 (2011).
43
T Yamashita, et al., Blockade of interleukin-6 signaling aggravates ischemic cerebral damage in mice: Possible involvement of Stat3 activation in the protection of neurons. J Neurochem 94, 459–468 (2005).
44
JV Alvarez, DA Frank, Genome-wide analysis of STAT target genes: Elucidating the mechanism of STAT-mediated oncogenesis. Cancer Biol Ther 3, 1045–1050 (2004).
45
RL Carpenter, HW Lo, STAT3 Target Genes Relevant to Human Cancers. Cancers (Basel) 6, 897–925 (2014).
46
G Niu, et al., Role of Stat3 in regulating p53 expression and function. Mol Cell Biol 25, 7432–7440 (2005).
47
N Arbour, et al., Mcl-1 is a key regulator of apoptosis during CNS development and after DNA damage. J Neurosci 28, 6068–6078 (2008).
48
MM Magiera, et al., Trim17-mediated ubiquitination and degradation of Mcl-1 initiate apoptosis in neurons. Cell Death Differ 20, 281–292 (2013).
49
Z Yuan, et al., Opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis. Mol Cell Biol 29, 2431–2442 (2009).
50
P Eversole-Cire, et al., Synergistic effect of Bcl-2 and BAG-1 on the prevention of photoreceptor cell death. Invest Ophthalmol Vis Sci 41, 1953–1961 (2000).
51
SH Tsang, et al., Retarding photoreceptor degeneration in Pdegtm1/Pdegtml mice by an apoptosis suppressor gene. Invest Ophthalmol Vis Sci 38, 943–950 (1997).
52
J Yang, GR Stark, Roles of unphosphorylated STATs in signaling. Cell Res 18, 443–451 (2008).
53
J Yang, et al., Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB. Genes Dev 21, 1396–1408 (2007).
54
H Tokami, et al., RANTES has a potential to play a neuroprotective role in an autocrine/paracrine manner after ischemic stroke. Brain Res; REBIOS Investigators 1517, 122–132 (2013).
55
J Wegrzyn, et al., Function of mitochondrial Stat3 in cellular respiration. Science 323, 793–797 (2009).
56
Q Zhang, et al., Mitochondrial localized Stat3 promotes breast cancer growth via phosphorylation of serine 727. J Biol Chem 288, 31280–31288 (2013).
57
D Lau, et al., Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2. Invest Ophthalmol Vis Sci 41, 3622–3633 (2000).
58
M Neuner-Jehle, et al., Ocular cell transfection with the human basic fibroblast growth factor gene delays photoreceptor cell degeneration in RCS rats. Hum Gene Ther 11, 1875–1890 (2000).
59
YH Huang, et al., STAT1 activation by venous malformations mutant Tie2-R849W antagonizes VEGF-A-mediated angiogenic response partly via reduced bFGF production. Angiogenesis 16, 207–222 (2013).
60
L Chen, W Wu, T Dentchev, R Wong, JL Dunaief, Increased metallothionein in light damaged mouse retinas. Exp Eye Res 79, 287–293 (2004).
61
KA Wunderlich, T Leveillard, M Penkowa, E Zrenner, MT Perez, Altered expression of metallothionein-I and -II and their receptor megalin in inherited photoreceptor degeneration. Invest Ophthalmol Vis Sci 51, 4809–4820 (2010).
62
K Tsuruma, et al., Metallothionein-III deficiency exacerbates light-induced retinal degeneration. Invest Ophthalmol Vis Sci 53, 7896–7903 (2012).
Information & Authors
Information
Published in
Classifications
Submission history
Published online: December 15, 2014
Published in issue: December 30, 2014
Keywords
Acknowledgments
We thank Dr. T. Li (Harvard University) for providing Tg(RHO P347S) mice, Dr. C. J. Watson (University of Cambridge) for providing Stat3fl/fl mice, Dr. C. K. Chen (Virginia Commonwealth University) for providing Tg(Opsin-iCre) mice, and Dr. K. Rajewsky (Harvard University) for providing R26+/LSL-Stat3C mice. We also thank the Montreal Genome Centre for performing microarray analyses and the Toronto Center for Phenogenomics for generating R26+/LSL-Stat3wt and R26+/LSL-Stat3Y705F mice. This work was supported by an award from the Vision Science Research Program provided by the Ontario Student Opportunity Trust Fund (to K.J.) and by Grants MOP-7315 and IOP-54037 (to R.R.M.) from the Canadian Institutes of Health Research, the Canadian Genetic Diseases Network, and the Macula Vision Research Foundation. R.R.M. is a Tier 1 Canada Research Chair in Neurogenetics. The S.E.E. laboratory was supported by The Terry Fox Foundation and the Canadian Breast Cancer Foundation. The rAAV8(Y733F)-hGRK1-Stat3-FLAG virus was generated by the W.W.H. laboratory. W.W.H. acknowledges partial support of this work from NIH Grants P30EY021721 and R01EY17549 and grants from the Macular Vision Research Foundation; Overstreet Fund; and Research to Prevent Blindness, Inc. The subretinal injection and ERGs were performed by the Q.L. laboratory, supported, in part, by NIH Grant EY021752; the Overstreet Fund; and Research to Prevent Blindness, Inc.
Notes
This article is a PNAS Direct Submission.
Authors
Competing Interests
Conflict of interest statement: W.W.H. and the University of Florida have a financial interest in the use of adenoassociated virus therapies, and own equity in a company (AGTC, Inc.) that might, in the future, commercialize some aspects of this work.
Metrics & Citations
Metrics
Citation statements
Altmetrics
Citations
If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.