Markus Thiersch

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)

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.

HIF1A is essential for the development of the intermediate plexus of the retinal vasculature.

PURPOSE HIF1A is one of the major transcription factors that regulate tissue response to low oxygen tension. It controls expression of a large number of genes involved in cell survival, proliferation, angiogenesis, and other cellular processes. HIF1A is present at increased levels in the early postnatal retina. In this study its potential function during postnatal development of the mouse retina and retinal vasculature was analyzed. METHODS A mouse line was generated with a Cre-mediated Hif1a knockdown in the peripheral retina. Retinal morphology and vasculature were analyzed in sections and flat mount preparations. Gene and protein expression were determined by real-time PCR and Western blot analysis. RESULTS The Cre-mediated knockdown caused a significant reduction in Hif1a gene expression and HIF1A protein levels in the early postnatal retina. Retinal morphology was normal but the Hif1a knockdown prevented the formation of the intermediate vascular plexus in the peripheral retina. The primary plexus and the outer plexus were less affected. The Hif1a knockdown did not affect expression of such angiogenesis-related genes as vascular endothelial growth factor (Vegf) but strongly induced expression of erythropoietin (Epo). At the protein level, EPAS1 (HIF2A) was stabilized in the Hif1a knockdown mice. CONCLUSIONS The results suggest that HIF1A may be directly or indirectly required for normal development of the retinal vasculature, especially of the intermediate plexus. EPO but not VEGF may play a significant role in the development of this phenotype. HIF1A may not be the main factor that regulates Vegf expression during retinal development in the mouse.

Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia‐inducible factor‐1α expression in photoreceptors

Hypoxic preconditioning stabilizes hypoxia‐inducible factor (HIF) 1α in the retina and protects photoreceptors against light‐induced cell death. HIF‐1α is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor‐specific expression of HIF‐1α is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF‐1α specifically in photoreceptor cells, using the cyclization recombinase (Cre)–lox system. The Cre‐mediated knockdown caused a 20‐fold reduced expression of Hif‐1α in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF‐1α protein levels. Accordingly, HIF‐1 target gene expression after hypoxia was significantly diminished. Retinas of Hif‐1α knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild‐type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light‐induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia‐mediated survival of light exposure does not depend on an autocrine action of HIF‐1α in photoreceptor cells. Hypoxia‐mediated stabilization of HIF‐2α and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif‐1α knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

Normoxic activation of hypoxia-inducible factors in photoreceptors provides transient protection against light-induced retinal degeneration.

PURPOSE Hypoxic preconditioning activates hypoxia-inducible transcription factors (HIFs) in the retina and protects photoreceptors from light-induced retinal degeneration. The authors tested whether photoreceptor-specific activation of HIFs in normoxia is sufficient for protection. METHODS Rod-specific Vhl knockdown mice were generated using the Cre-lox system with the rod opsin promoter controlling expression of CRE recombinase to stabilize HIF transcription factors in normoxic rods. Cell death was induced by light exposure and quantified by ELISA. Rhodopsin was quantified by spectrophotometry. Gene expression was analyzed by real-time PCR, and levels of proteins were determined by Western blotting. Morphology was investigated by light microscopy and retinal function tested by ERG. RESULTS The rod-specific Vhl knockdown stabilized HIF-α proteins and induced expression of HIF target genes in retinas of 10-week-old mice under normoxic conditions. Retinal morphology and function were normal. At 36 hours after exposure to excessive light, Vhl knockdowns showed significantly less photoreceptor cell death than did wild-type controls. Ten days after light exposure, however, photoreceptor degeneration in Vhl knockdowns was similar to that of control animals. Vhl knockdowns expressed Fgf2 at higher basal levels before light exposure. After light exposure, however, expression of Fgf2 was not significantly different from that of wild-type controls. CONCLUSIONS Artificial activation of HIF transcription factors in normoxic photoreceptors results in an increased basal expression of Fgf2 that may contribute to a transient protection of rods against light damage. Full photoreceptor protection may require a hypoxia-like response in additional retinal cell types and/or the differential regulation of additional mechanisms.

Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation

Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

The Hypoxic Transcriptome of the Retina: Identification of Factors with Potential Neuroprotective Activity

Thiersch, M; Raffelsberger, W; Frigg, E; Samardzija, M; Blank, P; Poch, O; Grimm, C Thiersch, M; Raffelsberger, W; Frigg, E; Samardzija, M; Blank, P; Poch, O; Grimm, C (2008). The hypoxic transcriptome of the retina: identification of factors with potential neuroprotective activity. In: Anderson, R E [et al.]. Recent Advances in Retinal Degeneration. New York, 75-85. Postprint available at: http://www.zora.uzh.ch

The differential role of Jak/STAT signaling in retinal degeneration.

Retinal degenerative diseases are a major cause of severe visual impairment or blindness in humans. To develop therapeutic strategies it is of particular importance to understand the molecular mechanisms taking place during the progression of the disease. Genes and proteins of the Janus kinase/Signal Transducer and Activator of Transcription (Jak/STAT) signaling pathway have been shown to play an important role in models of retinal degeneration (RD). Here we investigated the expression of additional genes involved in the Jak/STAT pathway in an induced (light exposure) and an inherited (rd1 mouse) model of RD. We show that STAT mRNAs as well as the Jak2/shp-1 pathway are differentially regulated in the two models. In contrast, we show that Jak3 mRNA is upregulated in both, the light damaged and the degenerative retina of the rd1 mouse. This common answer to probably different apoptotic stimuli suggests a prominent role for Jak3 in the damaged retina and could therefore be interesting for further investigations.

LIF-dependent JAK3 activation is not essential for retinal degeneration.

J. Neurochem. (2010) 113, 1210–1220.