Marijana Samardzija

HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1α (HIF-1α) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood–retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

In vivo confocal imaging of the retina in animal models using scanning laser ophthalmoscopy

Scanning-laser ophthalmoscopy is a technique for confocal imaging of the eye in vivo. The use of lasers of different wavelengths allows to obtain information about specific tissues and layers due to their reflection and transmission characteristics. In addition, fluorescent dyes excitable in the blue and infrared range offer a unique access to the vascular structures associated with each layer. In animal models, a further enhancement in specificity can be obtained by GFP expression under control of tissue-specific promotors. Important fields of application are studies in retinal degenerations and the follow-up of therapeutic intervention.

Constitutive Overexpression of Human Erythropoietin Protects the Mouse Retina against Induced But Not Inherited Retinal Degeneration

Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments.

Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

Leukemia Inhibitory Factor Extends the Lifespan of Injured Photoreceptors In Vivo

Survival and death of photoreceptors in degenerative diseases of the retina is controlled by a multitude of genes and endogenous factors. Some genes may be involved in the degenerative process itself whereas others may be part of an endogenous defense system. We show in two models of retinal degeneration that photoreceptor death strongly induces expression of leukemia inhibitory factor (LIF) in a subset of Muller glia cells in the inner nuclear layer of the retina. LIF expression is essential to induce an extensive intraretinal signaling system which includes Muller cells and photoreceptors and is characterized by an upregulation of Edn2, STAT3, FGF2 and GFAP. In the absence of LIF, Muller cells remain quiescent, the signaling system is not activated and retinal degeneration is strongly accelerated. Intravitreal application of recombinant LIF induces the full molecular pathway including the activation of Muller cells in wild-type and Lif –/– mice. Interruption of the signaling cascade by an Edn2 receptor antagonist increases whereas activation of the receptor decreases photoreceptor cell death. Thus, LIF is essential and sufficient to activate an extensive molecular defense response to photoreceptor injury. Our data establish LIF as a Muller cell derived neuronal survival factor which controls an intrinsic protective mechanism that includes Edn2 signaling to support photoreceptor cell survival and to preserve vision in the injured retina.

Differential role of Jak-STAT signaling in retinal degenerations

Retinal degeneration is a major cause of severe visual impairment or blindness. Understanding the underlying molecular mechanisms is a prerequisite to develop therapeutic approaches for human patients. We show in three mouse models that induced and inherited retinal degeneration induces LIF and CLC as members of the interleukin (IL)‐6 family of proteins, activates proteins of the Jak‐STAT signaling pathway, and up‐regulates suppressors of cytokine signaling as a negative feedback loop. Inhibition of Jak2 leads to protection of photoreceptors in a model of induced but not in a model of inherited retinal degeneration. Differential activation of Akt suggests alternative pathways for cell death and/or survival in different models. Proteins induced during photoreceptor degeneration are not mainly expressed in photoreceptors but in cells of other retinal layers. This suggests a model in which photoreceptor injury is signaled to cells of the inner retina, which in turn initiate a response either to support viability or accelerate death of injured cells.—Samardzija, M., Wenzel, A., Aufenberg, S., Thiersch, M., Remé, C., Grimm, C. Differential role of Jak‐STAT signaling in retinal degenerations. FASEB J. 20, E1790 –E1801 (2006)

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light.

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr-/- mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the “classical” visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.

Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection

BackgroundRetinal degeneration is a main cause of blindness in humans. Neuroprotective therapies may be used to rescue retinal cells and preserve vision. Hypoxic preconditioning stabilizes the transcription factor HIF-1α in the retina and strongly protects photoreceptors in an animal model of light-induced retinal degeneration. To address the molecular mechanisms of the protection, we analyzed the transcriptome of the hypoxic retina using microarrays and real-time PCR.ResultsHypoxic exposure induced a marked alteration in the retinal transcriptome with significantly different expression levels of 431 genes immediately after hypoxic exposure. The normal expression profile was restored within 16 hours of reoxygenation. Among the differentially regulated genes, several candidates for neuroprotection were identified like metallothionein-1 and -2, the HIF-1 target gene adrenomedullin and the gene encoding the antioxidative and cytoprotective enzyme paraoxonase 1 which was previously not known to be a hypoxia responsive gene in the retina. The strongly upregulated cyclin dependent kinase inhibitor p21 was excluded from being essential for neuroprotection.ConclusionOur data suggest that neuroprotection after hypoxic preconditioning is the result of the differential expression of a multitude of genes which may act in concert to protect visual cells against a toxic insult.

Rpe65 as a modifier gene for inherited retinal degeneration

Light accelerates progression of retinal degeneration in many animal models of retinitis pigmentosa (RP). A sequence variant in the Rpe65 gene (Rpe65450Leu or Rpe65450Met) can act as a modulator of light‐damage susceptibility in mice by influencing the kinetics of rhodopsin regeneration and thus by modulating the photon absorption. Depending on exposure duration and light intensity applied, white fluorescent light induces photoreceptor apoptosis and retinal degeneration in wild‐type mice by the activation of one of two known molecular pathways. These pathways depend, respectively, on activation of the transcription factor c‐Fos/AP‐1 and on phototransduction activity. Here we tested Rpe65 as a genetic modifier for inherited retinal degeneration and analysed which degenerative pathway is activated in a transgenic mouse model of autosomal dominant RP. We show that retinal degeneration was reduced in mice expressing the Rpe65450Met variant and that these mice retained more visual pigment rhodopsin than did transgenic mice expressing the Rpe65450Leu variant. In addition, lack of phototransduction slowed retinal degeneration whereas ablation of c‐Fos had no effect. We conclude that sequence variations in the Rpe65 gene can act as genetic modifiers in inherited retinal degeneration, presumably by regulating the daily rate of photon absorption through the modulation of rhodopsin regeneration kinetics. Increased absorption of photons and/or light sensitivity appear to accelerate retinal degeneration via an apoptotic cascade which involves phototransduction but not c‐Fos.

PAX6-positive Müller glia cells express cell cycle markers but do not proliferate after photoreceptor injury in the mouse retina

In lower vertebrates, such as fish, Müller glia plays an essential role in the restoration of visual function after retinal degeneration by transdifferentiating into photoreceptors and other retinal neurons. During this process, Müller cells re‐enter the cell cycle, proliferate, and migrate from the inner nuclear layer (INL) to the photoreceptor layer where they express photoreceptor‐specific markers. This process of Müller cell transdifferentiation is absent in mammals, and the loss of photoreceptors leads to permanent vision deficits. The mechanisms underlying the failure of mammalian Müller cells to behave as stem cells after photoreceptor degeneration are poorly understood. In the present study, we show that photoreceptor injury induces migration of PAX6‐positive Müller cell nuclei toward the outer part of the INL and into the inner part of the outer nuclear layer. These cells express markers of the cell cycle, suggesting an attempt to re‐enter the cell cycle similarly to lower vertebrates. However, mouse Müller cells do not proliferate in response to photoreceptor injury implying a blockade of the S‐phase transition. Our results suggest that a release of the S‐phase blockade may be crucial for Müller cells to successfully transdifferentiate and replace injured photoreceptors in mammals.