STAT3 promotes survival of mutant photoreceptors in inherited photoreceptor degeneration models

Ke Jiang; Katherine L. Wright; Ping Zhu; Michael J. Szego; Alexa N. Bramall; William W. Hauswirth; Qiuhong Li; Sean E. Egan; Roderick R. McInnes

doi:10.1073/pnas.1411248112

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 Prph2^rds^/+ 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 (Stat3^wt) or the gain-of-function variant STAT3^C (Stat3^C) improved PR survival in both models. Moreover, PR signaling in Tg(RHO P347S) mice carrying either a R26-Stat3^wt or R26-Stat3^C 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-Stat3^Y705F 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.

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 Prph2^rds/+ 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 Prph2^rds/+ 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 STAT3^Tyr705 (pSTAT3^Tyr705) 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 pSTAT3^Tyr705 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 pSTAT3^Tyr705 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/pSTAT3^Tyr705 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 Prph2^rds/+ 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 Prph2^rds/+ Mice.

To identify differentially expressed genes in IPDs, we analyzed gene expression in retinas from two IPD models, Tg(RHO P347S) and Prph2^rds/+ 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 Prph2^rds/+ 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 pSTAT3^Tyr705 to be increased in both IPD models vs. age-matched WT controls: STAT3 and pSTAT3^Tyr705 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 Prph2^rds/+ retinas, respectively, at 7 wk (Fig. 1A).

Fig. 1.

Stat3 expression is increased in Müller glia and mutant PRs of Tg(RHO P347S) and Prph2^rds/+ mice. (A) Immunoblot analyses of STAT3 and pSTAT3^Tyr705 in whole retinal protein extracts. Total retinal protein from Tg(RHO P347S) retinas (n = 3) at PN21, Prph2^rds/+ retinas (n = 3) at 7 wk of age, and age-matched WT controls (n = 3 for each group) were used to compare STAT3 and pSTAT3^Tyr705 expression. β-actin and CHX10 were loading controls. (B) IF staining of STAT3 and pSTAT3^Tyr705 on paraffin sections of Tg(RHO P347S) retinas at PN21, Prph2^rds/+ retinas at 7 wk, and age-matched WT controls. p21^kip1 is a Müller glia nucleus marker. (Scale bars: 25 μm.) (C) qPCR analysis on laser-captured INL and ONL samples from Tg(RHO P347S) retinas at PN21, Prph2^rds/+ retinas at 7 wk, and age-matched WT controls (n = 3 for all samples). Chx10 is an INL marker, and Rho is an ONL marker. Two-tailed Student t tests were used in all data analyses. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Stat3 Expression Is Increased in Both Müller Glia and Mutant PRs in Tg(RHO P347S) and Prph2^rds/+ Mice.

To define retinal cell types expressing the increased levels of STAT3 and pSTAT3^Tyr705, we performed immunofluorescence (IF) staining on Tg(RHO P347S) retinas at PN21, on Prph2^rds/+ 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 pSTAT3^Tyr705 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 pSTAT3^Tyr705 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 Prph2^rds/+ 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 Prph2^rds/+ 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 Prph2^rds/+ 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 Prph2^rds/+ 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 Prph2^rds/+ mice. To this end, we first introduced homozygous Stat3^fl/fl alleles (28) into both Tg(RHO P347S) and Prph2^rds/+ 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.

PR-specific deletion of Stat3 decreased PR survival in both Tg(RHO P347S) and Prph2^rds/+ mice. (A) Histology and thickness of the ONL were comparable in toluidine blue-stained Tg(Opsin-iCre) retinas vs. WT retinas (n = 5; P = 0.59) at PN40. (B) ONL thickness was not different between Stat3^fl/fl and Stat3^fl/fl; Tg(Opsin-iCre) retinas (n = 6; P = 0.86) at PN40. (C) ONL thickness was not different between Tg(RHO P347S) and Tg(RHO P347S); Tg(Opsin-iCre) retinas (n = 7; P = 0.82) at PN30. Deletion of Stat3 in Tg(RHO P347S) PRs resulted in a mean decrease of 40% in ONL thickness at PN20 (D, n = 6) and 67% at PN30 (E, n = 5) vs. littermate controls. (Scale bar: 25 μm.) (F) Bar graph of ONL thickness for each group in A–E. (G) Histology and thickness of the ONL were comparable in toluidine blue-stained Tg(Opsin-iCre) retinas vs. WT retinas (n = 8; P = 0.88) at 6 mo. (H) ONL thickness was not different between Stat3^fl/fl and Stat3^fl/fl; Tg(Opsin-iCre) retinas (n = 4; P = 0.36) at 6 mo. (I) ONL thickness was not different between Prph2^rds/+ and Prph2^rds/+; Tg(Opsin-iCre) retinas (n = 13; P = 0.27) at 6 mo. Deletion of Stat3 in Prph2^rds/+ PRs resulted in a mean decrease of 17% in ONL thickness at 2 mo (J, n = 7) and 50% at 6 mo (K, n = 9) vs. littermate controls. (Scale bar: 25 μm.) (L) Bar graph of ONL thickness for each group in G–K. Two-tailed Student t tests were used for all data analyses. Data are shown as mean ± SEM. **P < 0.01; ***P < 0.001.

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 Stat3^fl/fl; Tg(RHO P347S); Tg(Opsin-iCre) retinas at PN20 and from the PRs of Stat3^fl/fl; Prph2^rds/+; 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 Stat3^fl/fl and Stat3^fl/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 Prph2^rds/+; Tg(Opsin-iCre) and Prph2^rds/+ 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 Stat3^fl/fl; Tg(RHO P347S); Tg(Opsin-iCre) vs. Stat3^fl/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 Stat3^fl/fl; Prph2^rds/+; Tg(Opsin-iCre) vs. Stat3^fl/fl; Prph2^rds/+ 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 (Stat3^wt), a gain-of-function (Stat3^C) (33), or a dominant negative (Stat3^Y705F) allele, specifically in PRs (Fig. S4). The Cys residues in STAT3^C (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, STAT3^Y705F 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 Prph2^rds/+; 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 Stat3^wt 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 Stat3^C, 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 Stat3^Y705F 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 Prph2^rds/+ mice. At 6 mo, ONL thickness in Prph2^rds/+ retinas was increased by 14% (n = 10; P < 0.05) with expression of the Stat3^wt allele (Fig. 3 G and J) and by 19% (n = 7; P < 0.001) with expression of the Stat3^C allele (Fig. 3 H and J), whereas expression of the Stat3^Y705F 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.

PR-specific expression of a Stat3^wt or Stat3^C allele from the R26 locus increased PR survival in both Tg(RHO P347S) and Prph2^rds/+ mice. (A) Histology and thickness of the ONL at PN40 were comparable in toluidine blue-stained R26^{+/LSL-Stat3wt} vs. R26^{+/LSL-Stat3wt}; Tg(Opsin-iCre) retinas (n = 7; P = 0.63). At PN30, expression of either a Stat3^wt allele (B) or a Stat3^C allele (C) in Tg(RHO P347S) PRs increased the mean ONL thickness by 26% (n = 14) or 39% (n = 12), respectively, vs. littermate controls. (D) ONL thickness at PN30 was not significantly different from the expression of a Stat3^Y705F allele in Tg(RHO P347S) PRs (n = 13; P = 0.61). (Scale bar: 25 μm.) (E) Bar graph of ONL thickness for each group in A–D. Data from R26^{+/LSL-Stat3wt} (n = 7), R26^+/LSL-Stat3C (n = 8), and R26^{+/LSL-Stat3Y705F} (n = 7) retinas were combined and presented as R26-Stat3. (F) Histology and thickness of the ONL at 6 mo were comparable in toluidine blue-stained R26^{+/LSL-Stat3wt} vs. R26^{+/LSL-Stat3wt}; Tg(Opsin-iCre) retinas (n = 3; P = 0.80). At 6 mo, expression of either a Stat3^wt allele (G) or a Stat3^C allele (H) in Prph2^rds/+ PRs increased the mean ONL thickness by 14% (n = 10) and 19% (n = 7), respectively, vs. littermate controls. (I) ONL thickness at 6 mo was not significantly different from the expression of a Stat3^Y705F allele in Prph2^rds/+ PRs (n = 5; P = 0.53). (Scale bar: 25 μm.) (J) Bar graph of ONL thickness of each group in F–I. Data from R26^{+/LSL-Stat3wt} (n = 3), R26^+/LSL-Stat3C (n = 3), and R26^{+/LSL-Stat3Y705F} (n = 3) retinas were combined and presented as R26-Stat3. Two-tailed Student t tests were used in all data analyses. Data are shown as mean ± SEM. *P < 0.05; ***P < 0.001.

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 Stat3^wt allele at PN30 (n = 10; P < 0.05) or the Stat3^C 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-specific expression of the R26-Stat3^wt or R26-Stat3^C allele in Tg(RHO P347S) mice increased the ERG a-wave. Scotopic responses were measured in dark-adapted mice at 2.5 cd⋅s⋅m⁻². No difference in a-wave amplitude was seen between R26^{+/LSL-Stat3wt} and R26^{+/LSL-Stat3wt}; Tg(Opsin-iCre) mice at PN20 (n = 4; P = 0.66), PN30 (n = 6; P = 0.17), or PN45 (n = 5; P = 0.19). Expression of a Stat3^wt allele in the PRs did not alter the a-wave (n = 9; P = 0.70) amplitudes of Tg(RHO P347S) mice at PN20. In contrast, a significant increase in a-wave amplitude was observed in R26^{+/LSL-Stat3wt}; Tg(RHO P347S); Tg(Opsin-iCre) mice at PN30 (n = 10; P < 0.05) vs. R26^{+/LSL-Stat3wt}; Tg(RHO P347S) mice. Expression of the Stat3^C allele in Tg(RHO P347S) PRs significantly improved a-wave amplitude at PN20 (n = 16; P < 0.05) and PN30 (n = 15; P < 0.001). No differences in a-wave amplitudes were observed in Tg(RHO P347S) mice at PN45 with expression of either the Stat3^wt or Stat3^C allele. Two-tailed Student t tests were used in all data analyses. *P < 0.05; ***P < 0.001.

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.

Subretinal injection of rAAV8(Y733F)-hGRK1-Stat3-FLAG increased PR survival in Tg(RHO P347S) mice. Tg(RHO P347S) mice were injected subretinally in one eye with rAAV8(Y733F)-hGRK1-Stat3-FLAG on PN14, while leaving the other eye intact. (A) IF staining of FLAG was used at PN60 to detect expression of STAT3-FLAG protein on paraffin sections of WT retinas uninjected or subretinally injected with rAAV8(Y733F)-hGRK1-Stat3-FLAG. IS, inner segment; OS, outer segment. (Scale bar: 25 μm.) (B) Representative toluidine blue-stained cross-sections of Tg(RHO P347S) retinas at PN60 from the injected and uninjected eyes of a single representative animal. (C) ONL thickness at PN60 of both retinas in each Tg(RHO P347S) mouse. (D) Percentage change in ONL thickness at PN60, comparing the rAAV8(Y733F)-hGRK1-Stat3-FLAG–injected retina with the uninjected retina of each Tg(RHO P347S) mouse. A mean increase in ONL thickness of 15 ± 5% (n = 18; P < 0.01) was seen with rAAV8(Y733F)-hGRK1-Stat3-FLAG injection. (E) Percentage change in scotopic a-wave amplitude in each Tg(RHO P347S) mouse at PN60, under different light intensities, comparing the injected retina and the uninjected retina. The bar boxes show the mean for each group (n = 7 or more for each group). Paired two-tailed Student t tests were used in all data analyses. Data are shown as mean ± SD. **P < 0.01; ***P < 0.001.

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 pSTAT3^Tyr705 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 Prph2^rds/+ mice. Second, deletion of Stat3 in mutant PRs greatly increased the degree of PR death. Third, augmentation of either STAT3^wt or STAT3^C expression, using R26 transgenes, significantly enhanced mutant PR survival in both Tg(RHO P347S) and Prph2^rds/+ 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 pSTAT3^Tyr705 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 pSTAT3^Tyr705 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 pSTAT3^Tyr705 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. pSTAT3^Tyr705 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 pSTAT3^Tyr705 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 pSTAT3^Tyr705 may also be mediated by an increase in the abundance of unphosphorylated STAT3 (U-STAT3): Stat3 expression is activated by pSTAT3^Tyr705 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, pStat3^Ser727, may also play a significant role in IPD pathogenesis. pStat3^Ser727 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 pSTAT3^Tyr705 binds directly to the FGF2 promoter (59), further suggesting that as a putative pSTAT3^Tyr705 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.

Prph2^rds/rds (C3A.BLiA-Pde6b⁺.O20-Prph2^Rd2/J) mice, their corresponding control stain Prph2^+/+ (C3A.BLiA-Pde6b⁺/J), and C57BL/6J mice were obtained from the Jackson Laboratory. Prph2^rds/+ mice were generated by crossing Prph2^rds/rds and Prph2^+/+ mice. Tg(RHO P347S) (C57BL/6J) mice were provided by Tiansen Li, Harvard University, Boston, MA, Stat3^flox/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-pSTAT3^Tyr705 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, Prph2^rds/+, 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-pSTAT3^Tyr705 at a 1:100 dilution (Cell Signaling), anti-p27^kip1 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, pSTAT3^Tyr705, Cre, and FLAG in a 1:100 dilution of goat anti-rabbit Cy3 (Invitrogen) was used, whereas for p27^kip1, 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 Stat3^fl/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.

Footnotes

↵¹To whom correspondence may be addressed. Email: segan{at}sickkids.ca or rod.mcinnes{at}mcgill.ca.

Author contributions: K.J., Q.L., S.E.E., and R.R.M. designed research; K.J., K.L.W., P.Z., M.J.S., and A.N.B. performed research; K.L.W., W.W.H., Q.L., and S.E.E. contributed new reagents/analytic tools; K.J. analyzed data; and K.J., S.E.E., and R.R.M. wrote the paper.
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.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1411248112/-/DCSupplemental.

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[1] ↵
Bramall AN,
Wright AF,
Jacobson SG,
McInnes RR
(2010) 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
.
OpenUrl CrossRef PubMed

[2] Bramall AN,

[3] Wright AF,

[4] Jacobson SG,

[5] McInnes RR

[6] ↵
Wright AF,
Chakarova CF,
Abd El-Aziz MM,
Bhattacharya SS
(2010) Photoreceptor degeneration: Genetic and mechanistic dissection of a complex trait. Nat Rev Genet 11(4):273–284
.
OpenUrl CrossRef PubMed

[7] Wright AF,

[8] Chakarova CF,

[9] Abd El-Aziz MM,

[10] Bhattacharya SS

[11] ↵
Rattner A,
Nathans J
(2005) The genomic response to retinal disease and injury: Evidence for endothelin signaling from photoreceptors to glia. J Neurosci 25(18):4540–4549
.
OpenUrl Abstract/FREE Full Text

[12] Rattner A,

[13] Nathans J

[14] ↵
Bramall AN, et al.
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STAT3 promotes survival of mutant photoreceptors in inherited photoreceptor degeneration models

Significance

Abstract

Results

Stat3 Expression Is Elevated in Retinas from Tg(RHO P347S) and Prph2^rds/+ Mice.

Stat3 Expression Is Increased in Both Müller Glia and Mutant PRs in Tg(RHO P347S) and Prph2^rds/+ Mice.

PR-Specific Deletion of Stat3 Decreases PR Survival in Both Tg(RHO P347S) and Prph2^rds/+ Mice.

Augmentation of PR Stat3 Expression from Rosa26-Stat3 Alleles Increases PR Survival.

PR Expression of the rAAV8(Y733F)-hGRK1-Stat3-FLAG Vector Increases PR Survival and Retinal Electrophysiological Responses in Tg(RHO P347S) Mice.

Discussion

Methods

Mice.

Immunoblotting.

Immunostaining.

Laser-Capture Microdissection.

Retinal ONL Measurements.

Electroretinography.

Subretinal Injection.

Statistical Analysis.

Acknowledgments

Footnotes

References

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Search

Significance

Abstract

Results

Stat3 Expression Is Elevated in Retinas from Tg(RHO P347S) and Prph2rds/+ Mice.

Stat3 Expression Is Increased in Both Müller Glia and Mutant PRs in Tg(RHO P347S) and Prph2rds/+ Mice.

PR-Specific Deletion of Stat3 Decreases PR Survival in Both Tg(RHO P347S) and Prph2rds/+ Mice.

Augmentation of PR Stat3 Expression from Rosa26-Stat3 Alleles Increases PR Survival.

PR Expression of the rAAV8(Y733F)-hGRK1-Stat3-FLAG Vector Increases PR Survival and Retinal Electrophysiological Responses in Tg(RHO P347S) Mice.

Discussion

Methods

Mice.

Immunoblotting.

Immunostaining.

Laser-Capture Microdissection.

Retinal ONL Measurements.

Electroretinography.

Subretinal Injection.

Statistical Analysis.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

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Information

Stat3 Expression Is Elevated in Retinas from Tg(RHO P347S) and Prph2^rds/+ Mice.

Stat3 Expression Is Increased in Both Müller Glia and Mutant PRs in Tg(RHO P347S) and Prph2^rds/+ Mice.

PR-Specific Deletion of Stat3 Decreases PR Survival in Both Tg(RHO P347S) and Prph2^rds/+ Mice.