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Differential role of Jak-STAT signaling in retinal degenerations

Laboratory for Retinal Cell Biology, Department Ophthalmology, Center for Integrative Human Physiology (CIHP) and Neuroscience Center Zurich (ZNZ), University Hospital, Zürich, Switzerland

¹Correspondence: Laboratory for Retinal Cell Biology, Department Ophthalmology, University Hospital, Frauenklinikstrasse 24, 8091 Zürich, Switzerland. E-mail: cgrimm{at}opht.unizh.ch

SPECIFIC AIMS

Photoreceptor apoptosis is a hallmark of blinding diseases caused by retinal degeneration. Neuroprotective strategies may protect photoreceptors from cell death and thus prolong the period of useful vision in patients. For the development of such strategies it is essential to understand the molecular signaling pathways involved in the degeneration of the retina. Molecules of the Jak-STAT pathway have been implicated in the control of cell survival, cell progression, and cell death. We analyzed a potential role of Jak-STAT signaling in three models of induced and inherited retinal degeneration. Using an inhibitor of Jak2, we tested whether the Jak-STAT pathway might be a potential target for therapeutic approaches, with the goal to prevent photoreceptor apoptosis.

PRINCIPAL FINDINGS

1. Induction of the Jak-STAT signaling pathway in a model of light-induced retinal degeneration
Retinal expression of leukemia inhibitory factor (LIF) and cardiotrophin like cytokine (CLC), members of the interleukin (IL)-6 family of cytokines, was strongly up-regulated during photoreceptor degeneration induced by exposure to bright white light. Peak expression of LIF and CLC occurred 12 h after light exposure, declining to near-basal levels in 3 days.

Concomitant with the induction of the cytokines, Janus kinase-2 (Jak2) as well as signal transducer and activator of transcription-1 (STAT1) and STAT3, both targets of Jak2, were activated by phosphorylation (Fig. 1 ). Extracellular-regulated kinase 1,2 (ERK1,2) was phosphorylated but Akt was not. This suggests that LIF and/or CLC were binding to their respective membrane receptors, leading to the activation of the intracellular Jak-STAT signaling pathway.

Jak2 and STAT-3 localized mainly to cells of the inner nuclear layer (INL) and the ganglion cell layer (GCL) with a weaker signal of STAT-3 in the inner segments of photoreceptors. STAT-1 localized mainly to the inner and outer plexiform layers (IPL and OPL) as well as to the GCL. This localization agrees with earlier publications and suggests that cells of the INL and GCL may respond to photoreceptor cell death.

The family of suppressors of cytokine signaling (SOCS) constitute a negative feedback loop for the regulation of Jak-STAT signaling. Expression analysis of all eight members of the SOCS family revealed a strong induction of SOCS3 (>13-fold) and, to a lesser extent, of SOCS1 (>6-fold) and of CIS (>2-fold).

2. Light-induced activation of the Jak-STAT pathway is damage dependent
To evaluate the significance of the activation of the Jak-STAT pathway for retinal degeneration, we tested phosphorylation of Jak2, STAT1, STAT3, and ERK1,2 in mouse strains differentially susceptible to light-induced retinal degeneration. Retinas of mice lacking the c-Fos protein on a genetic background expressing the methionine variant of the Rpe65 gene (c-fos^–/–; Rpe65_450M) are not susceptible to light damage whereas photoreceptors of c-fos^–/– mice expressing the leucine variant of the Rpe65 gene (Rpe65_450L) undergo rapid apoptosis after acute light exposure.

Phosphorylation of ERK1,2 was increased after exposure in all strains, suggesting that ERK1,2 is activated by light per se and so may be involved in a physiological response to increased lighting conditions. In contrast, Jak2, STAT1, and STAT3 were activated exclusively in strains that are susceptible to light damage. No activation could be detected in the resistant strain. Thus, the Jak-STAT signaling pathway may be important for the induction of cell death after the light stimulus, an interpretation supported by the peak of activation at 12 h postillumination, 24 h before the maximal release of free nucleosomes, which was analyzed as a biochemical marker for apoptosis.

3. Induction of members of the Jak-STAT pathway in inherited retinal degeneration
We used the rd1 mouse as a model for autosomal recessive retinitis pigmentosa (arRP) and the transgenic VPP mouse as a model for autosomal dominant RP (adRP) to test whether the pathway activated by acute light exposure may also be involved in photoreceptor apoptosis in inherited retinal degeneration. A mutation in the ßbeta;-subunit of phosphodiesterase causes the rapid degeneration of photoreceptors in the rd1 mouse, with an early onset around postnatal day (PND) 11 and almost complete degeneration at 3 wk of age. The transgene in the VPP mouse expresses a mutant rhodopsin protein with three amino acid substitutions. Degeneration induced by the transgene has a later onset and is slowly progressing. As in the light damage model, p-STAT3 levels were up-regulated during the phase of photoreceptor degeneration in both inherited models. Although levels of total STAT1 protein were elevated in both inherited models, p-STAT1 was not (rd1) or was only barely (VPP) detectable. In contrast to the induced model, p-Jak2 was not (rd1) or only marginally (VPP) activated, and levels of p-ERK1,2 remained constant. Instead, levels of p-Akt were elevated in the adult retinas of both models.

Exponential RT-polymerase chain reaction (RT-PCR) showed that LIF and CLC cytokines were also induced in both inherited models, as was SOCS3. CIS was slightly elevated in the VPP model but not in the rd1 mouse. SOCS1, however, remained at basal levels in both models. These results suggest that a degeneration-inducing stimulus causes a specific expression and/or activation of members of the Jak-STAT pathway. This is further supported by our observation that ciliary neurotrophic factor (CNTF) was moderately induced in the rd1 mouse retina and that fibroblast growth factor-2 (FGF-2) was strongly up-regulated in the VPP mouse but only marginally in the rd1 retina.

4. Inhibition of Jak-STAT signaling protects against induced but not inherited retinal degeneration
AG-490, an inhibitor of Jak2 activity, was injected i.p. immediately before the onset of light exposure. In contrast to control mice injected with carrier or ones that did not receive an injection, AG-490-treated mice did not show increased protein phosphorylation (Fig. 2 ). Furthermore, AG-490 treatment, and thus interference with the Jak-STAT pathway, significantly reduced light damage susceptibility of photoreceptors as indicated by reduced internucleosomal DNA cleavage (Fig. 2B ). AG-490-treated mice had only a few scattered photoreceptor nuclei showing condensed chromatin as a classical sign of apoptosis (Fig. 2C , middle panel), whereas mice treated with carrier only showed a highly increased number of pyknotic nuclei (Fig. 2C , bottom panel).

To assess the potential role of Jak2 in STAT3 activation and retinal degeneration in the VPP mouse, we treated VPP mice with daily injections of AG-490, starting at PND 15 and ending at PND 27. Analysis was at PND 28. The retina of 15-day-old VPP mice had 7 to 9 rows of photoreceptor nuclei and distinct (but shortened) inner and outer segments. AG-490 treatment reduced phosphorylation of Jak2 to levels found in wild-type (WT) animals. Levels of p-STAT3, however, were still elevated and clearly above WT levels. The amount of rhodopsin per retina was measured to quantify photoreceptor degeneration. Four-wk-old WT mice had 0.43 ± 0.083 (n=3) nmol rhodopsin, whereas untreated VPP mice of the same age had only 0.071 ± 0.015 (n=3) nmol and AG-490-treated mice had 0.065 ± 0.014 (n=4) rhodopsin left. Thus, regardless of the AG-490 treatment, >80% of the photoreceptors were lost.

CONCLUSIONS AND SIGNIFICANCE

In this work we show that members of the Jak-STAT pathway are induced in three different models of retinal degeneration. The individual activation patterns, however, are death stimulus specific. Differentially activated proteins include cytokine ligands, STAT transcription factors, and molecules of a negative feedback loop. The neuroprotective effect of the inhibition of Jak2 in induced but not in inherited cell death, supports the conclusion of different molecular pathways in retinal degenerations.

The Jak-STAT signaling pathway has been implicated in neurodegenerative as well as neuroprotective mechanisms. Activation of STAT1 is mostly proapoptotic whereas phosphorylation of STAT3 is commonly accepted as antiapoptotic. In the light-treated retina, STAT1 and STAT3 proteins were both activated, implying that cell death as well as cell survival mechanisms were induced. We speculate that the retina responds to an insult by generating both death factors promoting apoptosis of photoreceptors that are damaged beyond rescue and survival factors which help to rescue damaged photoreceptors that are still viable (Fig. 3 ). The production of such factors may be differentially controlled by STAT1 and STAT3, respectively. Jak2, STAT1, and STAT3 proteins were found mainly in cells of the INL and GCL and in the inner and outer plexiform layers. However, photoreceptors are the cells primarily affected in all models of retinal degeneration used here. Thus, the fate of photoreceptors might be determined by other cell types through STAT1/STAT3-regulated production and release of growth factors and/or cytokines.

Treatment with AG-490 did not rescue photoreceptors in the VPP mouse, which suggests that different mechanisms are involved in inherited retinal degenerations. The differential activation of STAT3 and STAT1 proteins, and especially the sustained phosphorylation of Akt in both inherited but not the induced model, support this conclusion. To find treatment strategies for a broad range of retinal degeneration, it is of outmost importance to analyze the different signaling cascades in order to identify common molecules as potential treatment targets.

View larger version (66K): [in this window] [in a new window]   Figure 1. Induction of the Jak-STAT pathway in the retina after acute light exposure. BALB/c mice were not (dark control, d) or were exposed to 5000 lux of white light for 1 h. After light exposure, mice were sacrificed immediately (i) or after two 96 h of recovery in darkness as indicated. Proteins were extracted from isolated retinas and tested by Western blot.

View larger version (111K): [in this window] [in a new window]   Figure 2. Inhibition of Jak signaling prevents retinal degeneration after bright light exposure. A) Immediately before light exposure, BALB/c mice received a single injection of 800 µg AG-490 (ag) or carrier DMSO (dm) or were not injected. Mice were subsequently exposed for 1 h to 5'000 lux or not (dark) and phosphorylation of proteins was tested 18 h postexposure. Retinal proteins from separate animals were loaded in each lane. B) Retinas of animals not exposed to light (dark) or of animals pretreated with AG-490 (ag) of DMSO (dm) and exposed for 1 h to 5'000 lux were used to quantify apoptosis by ELISA. *P = 0.003, 2-tailed t test; n = 10 mice for each condition. C) Light microscopy 18 h after light exposure. Retinas of dark control mice showed regular photoreceptors (upper panel). Retinas of AG-490 (ag)-treated mice exhibited few apoptotic photoreceptor nuclei (arrowheads, middle panel); retinas of carrier (DMSO, dm)-treated mice had significantly more dying photoreceptors (arrowheads, lower panel). RPE: retinal pigment epithelium; reactive oxygen species (ROS): rod outer segments; RIS: rod inner segments; ONL outer nuclear layer.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic representation of the signaling pathways involved in the models of retinal degeneration.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5895fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5895fjev1 20/13/2411    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Samardzija, M. Articles by Grimm, C. Search for Related Content PubMed PubMed Citation Articles by Samardzija, M. Articles by Grimm, C.