The extracellular matrix protein fibronectin promotes metanephric kidney development.

Complex interactions of the branching ureteric bud (UB) and surrounding mesenchymal cells during metanephric kidney development determine the final number of nephrons. Impaired nephron endowment predisposes to arterial hypertension and chronic kidney disease. In the kidney, extracellular matrix (ECM) proteins are usually regarded as acellular scaffolds or as the common histological end-point of chronic kidney diseases. Since only little is known about their physiological role in kidney development, we aimed for analyzing the expression and role of fibronectin. In mouse, fibronectin was expressed during all stages of kidney development with significant changes over time. At embryonic day (E) 12.5 and E13.5, fibronectin lined the UB epithelium, which became less pronounced at E16.5 and then switched to a glomerular expression in the postnatal and adult kidneys. Similar results were obtained in human kidneys. Deletion of fibronectin at E13.5 in cultured metanephric mouse kidneys resulted in reduced kidney sizes and impaired glomerulogenesis following reduced cell proliferation and branching of the UB epithelium. Fibronectin colocalized with alpha 8 integrin and fibronectin loss caused a reduction in alpha 8 integrin expression, release of glial-derived neurotrophic factor and expression of Wnt11, both of which are promoters of UB branching. In conclusion, the ECM protein fibronectin acts as a regulator of kidney development and is a determinant of the final nephron number.

Induced Attenuation of Scleral TGF-ß Signaling in Mutant Mice Increases Susceptibility to IOP-Induced Optic Nerve Damage.

Purpose: Axonal optic nerve (ON) damage in glaucoma is characteristically associated with increased amounts of active transforming growth factor-beta 2 (TGF-ß2) in the ON head. Here we investigated the functional role of scleral TGF-ß signaling in glaucoma. Methods: A deficiency of Tgfbr2, which encodes for TGF-ß receptor type II (TGF-ßRII), the essential receptor for canonical TGF-ß signaling, was induced in fibroblasts (including those of the sclera) of mutant mice. To this end, 5-week-old mice were treated with tamoxifen eye drops. Experimental glaucoma was induced in 8-week-old mice using a magnetic microbead (MB) model. After 6 weeks of high intraocular pressure (IOP), the ON axons and their somata in the retina were labeled by paraphenylenediamine (PPD) and RNA-binding protein with multiple splicing (RBPMS) immunohistochemistry, respectively, and quantified. Results: Tamoxifen treatment resulted in a significant decrease of TGF-ßRII and its mRNA in the sclera. After 6 weeks of high IOP, reduced numbers of PPD-stained ON axons were seen in MB-injected eyes in comparison with not-injected contralateral eyes. Moreover, MB injection also led to a decrease of retinal ganglion cell (RGC) somata as seen in RBPMS-stained retinal wholemounts. Axon loss and RGC loss were significantly higher in mice with a fibroblast specific deficiency of TGF-ßRII in comparison with control animals. Conclusions: We conclude that the ablation of scleral TGF-ß signaling increases the susceptibility to IOP-induced ON damage. Scleral TGF-ß signaling in mutant mice appears to be beneficial for ON axon survival in experimentally induced glaucoma.

Histomorphometry of the Sural Nerve for Use as a CFNG in Facial Reanimation Procedures.

Facial palsy (FP) is a debilitating nerve pathology. Cross Face Nerve Grafting (CFNG) describes a surgical technique that uses nerve grafts to reanimate the paralyzed face. The sural nerve has been shown to be a reliable nerve graft with little donor side morbidity. Therefore, we aimed to investigate the microanatomy of the sural nerve. Biopsies were obtained from 15 FP patients who underwent CFNG using sural nerve grafts. Histological cross-sections were fixated, stained with PPD, and digitized. Histomorphometry and a validated software-based axon quantification were conducted. The median age of the operated patients was 37 years (5-62 years). There was a significant difference in axonal capacity decrease towards the periphery when comparing proximal vs. distal biopsies (p = 0.047), while the side of nerve harvest showed no significant differences in nerve caliber (proximal p = 0.253, distal p = 0.506) and axonal capacity for proximal and distal biopsies (proximal p = 0.414, distal p = 0.922). Age did not correlate with axonal capacity (proximal: R = -0.201, p = 0.603; distal: R = 0.317, p = 0.292). These novel insights into the microanatomy of the sural nerve may help refine CFNG techniques and individualize FP patient treatment plans, ultimately improving overall patient outcomes.

Retinal Pigmented Epithelium-Derived Ectopic Norrin Does Not Promote Intraretinal Angiogenesis in Transgenic Mice.

Formation of intraretinal capillaries and inner blood-retinal barrier during development requires norrin, a ligand of the canonical wingless/integrated (Wnt)/ß-catenin signaling pathway. Here we addressed the question whether retinal pigmented epithelium (RPE)-derived overexpression of norrin in transgenic mice rescues the vascular phenotype caused by norrin deficiency. To this end, we generated NdpKO/Rpe65-Norrin mice and analyzed the activation of ß-catenin signaling, the development of intraretinal capillaries, and the expression of blood-retinal barrier marker molecules. RPE-derived norrin induced retinal ß-catenin signaling but failed to rescue the vascular developmental defects and the breakdown of the blood-retinal barrier in norrin-deficient mice. Sites of ectopic norrin expression and the amounts of secreted transgenic protein are critical factors to enable the angiogenic properties of norrin.

Intravenous injection of cyclosporin A loaded lipid nanocapsules fights inflammation and immune system activation in a mouse model of diabetic retinopathy.

Inflammation and immune system activation are key pathologic events in the onset and escalation of diabetic retinopathy (DR). Both are driven by cytokines and complement originating from the retinal pigment epithelium (RPE). Despite the RPE's pivotal role, there is no therapeutic tool to specifically interfere with the RPE-related pathomechanism. A therapy that addresses RPE cells and counteracts inflammation and immune response would be of paramount value for the early treatment of DR, where currently are no specific therapies available. Here, we utilized lipoprotein-mimetic lipid nanocapsules to deliver the anti-inflammatory and immunosuppressive drug cyclosporin A (CsA) to RPE cells. Using a mouse model of DR that mirrors all pathologic aspects of human DR, we demonstrate that intravenously applied CsA-loaded lipid nanocapsules comprehensively counteract inflammation and immune system activation. One single injection suppressed the expression of pro-inflammatory cytokines, dampened macrophage infiltration, and prevented macrophage and microglia activation in eyes with DR. This work shows that CsA-loaded lipid nanocapsules can offer new avenues for the treatment of DR.

A single intravenous injection of cyclosporin A-loaded lipid nanocapsules prevents retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a retinal disease that threatens the vision of prematurely born infants. Severe visual impairment up to complete blindness is caused by neovascularization and inflammation, progressively destroying the immature retina. ROP primarily affects newborns in middle- and low-income countries with limited access to current standard treatments such as intraocular drug injections and laser- or cryotherapy. To overcome these limitations, we developed a nanotherapeutic that effectively prevents ROP development with one simple intravenous injection. Its lipid nanocapsules transport the antiangiogenic and anti-inflammatory cyclosporin A efficiently into disease-driving retinal pigment epithelium cells. In a mouse model of ROP, a single intravenous injection of the nanotherapeutic prevented ROP and led to normal retinal development by counteracting neovascularization and inflammation. This nanotherapeutic approach has the potential to bring about a change of paradigm in ROP therapy and prevent millions of preterm born infants from developing ROP.

Targeted drug delivery to the retinal pigment epithelium: Untapped therapeutic potential for retinal diseases.

The retinal pigment epithelium (RPE) plays a crucial part in sight-threatening diseases. In this review, we shed light on the pivotal implication of the RPE in age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity; and explain why a paradigm shift toward targeted RPE therapy is needed to efficiently fight these retinal diseases. We provide guidance for the development of RPE-specific nanotherapeutics by giving a comprehensive overview of the possibilities and challenges of drug delivery to the RPE and highlight successful nanotherapeutic approaches targeting the RPE.

Deficiency in Retinal TGFß Signaling Aggravates Neurodegeneration by Modulating Pro-Apoptotic and MAP Kinase Pathways.

Transforming growth factor ß (TGFß) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFß receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFß signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFß signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFß signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFß signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.

Transcription factor Zfp276 drives oligodendroglial differentiation and myelination by switching off the progenitor cell program.

In oligodendrocytes of the vertebrate central nervous system a complex network of transcriptional regulators is required to ensure correct and timely myelination of neuronal axons. Here we identify Zfp276, the only mammalian ZAD-domain containing zinc finger protein, as a transcriptional regulator of oligodendrocyte differentiation and central myelination downstream of Sox10. In the central nervous system, Zfp276 is exclusively expressed in mature oligodendrocytes. Oligodendroglial deletion of Zfp276 led to strongly reduced expression of myelin genes in the early postnatal mouse spinal cord. Retroviral overexpression of Zfp276 in cultured oligodendrocyte precursor cells induced precocious expression of maturation markers and myelin genes, further supporting its role in oligodendroglial differentiation. On the molecular level, Zfp276 directly binds to and represses Sox10-dependent gene regulatory regions of immaturity factors and functionally interacts with the transcriptional repressor Zeb2 to enable fast transition of oligodendrocytes to the myelinating stage.

Role of the Pbrm1 subunit and the PBAF complex in Schwann cell development.

Myelin sheath formation in the peripheral nervous system and the ensuing saltatory conduction rely on differentiated Schwann cells. We have previously shown that transition of Schwann cells from an immature into a differentiated state requires Brg1 that serves as the central energy generating subunit in two related SWI/SNF-type chromatin remodelers, the BAF and the PBAF complex. Here we used conditional deletion of Pbrm1 to selectively interfere with the PBAF complex in Schwann cells. Despite efficient loss of Pbrm1 early during lineage progression, we failed to detect any substantial alterations in the number, proliferation or survival of immature Schwann cells as well as in their rate and timing of terminal differentiation. As a consequence, postnatal myelin formation in peripheral nerves appeared normal. There were no inflammatory alterations in the nerve or other signs of a peripheral neuropathy. We conclude from our study that Pbrm1 and very likely the PBAF complex are dispensable for proper Schwann cell development and that Schwann cell defects previously observed upon Brg1 deletion are mostly attributable to altered or absent function of the BAF complex.

Microanatomy of the Frontal Branch of the Facial Nerve: The Role of Nerve Caliber and Axonal Capacity.

BACKGROUND: A commonly seen issue in facial palsy patients is brow ptosis caused by paralysis of the frontalis muscle powered by the frontal branch of the facial nerve. Predominantly, static methods are used for correction. Functional restoration concepts include the transfer of the deep temporal branch of the trigeminal nerve and cross-facial nerve grafts. Both techniques can neurotize the original mimic muscles in early cases or power muscle transplants in late cases. Because axonal capacity is particularly important in cross-facial nerve graft procedures, the authors investigated the microanatomical features of the frontal branch to provide the basis for its potential use and to ease intraoperative donor nerve selection. METHODS: Nerve biopsy specimens from 106 fresh-frozen cadaver facial halves were obtained. Histologic processing and digitalization were followed by nerve morphometric analysis and semiautomated axon quantification. RESULTS: The frontal branch showed a median of three fascicles (n = 100; range, one to nine fascicles). A mean axonal capacity of 1191 ± 668 axons (range, 186 to 3539 axons; n = 88) and an average cross-sectional diameter of 1.01 ± 0.26 mm (range, 0.43 to 1.74 mm; n = 67) were noted. In the linear regression model, diameter and axonal capacity demonstrated a positive relation (n = 57; r2 = 0.32; p < 0.001). Based on that equation, a nerve measuring 1 mm is expected to carry 1339 axons. CONCLUSION: The authors' analysis on the microanatomy of the frontal branch could promote clinical use of cross-facial nerve graft procedures in frontalis muscle neurotization and free muscle transplantations.

Transcriptional Profiling Identifies Upregulation of Neuroprotective Pathways in Retinitis Pigmentosa.

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-ß regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-ß, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-ß, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

SoxD transcription factor deficiency in Schwann cells delays myelination in the developing peripheral nervous system.

The three SoxD proteins, Sox5, Sox6 and Sox13, represent closely related transcription factors with important roles during development. In the developing nervous system, SoxD proteins have so far been primarily studied in oligodendroglial cells and in interneurons of brain and spinal cord. In oligodendroglial cells, Sox5 and Sox6 jointly maintain the precursor state, interfere with terminal differentiation, and thereby ensure the proper timing of myelination in the central nervous system. Here we studied the role of SoxD proteins in Schwann cells, the functional counterpart of oligodendrocytes in the peripheral nervous system. We show that Schwann cells express Sox5 and Sox13 but not Sox6. Expression was transient and ceased with the onset of terminal differentiation. In mice with early Schwann cell-specific deletion of both Sox5 and Sox13, embryonic Schwann cell development was not substantially affected and progressed normally into the promyelinating stage. However, there was a mild and transient delay in the myelination of the peripheral nervous system of these mice. We therefore conclude that SoxD proteins-in stark contrast to their action in oligodendrocytes-promote differentiation and myelination in Schwann cells.

A novel ocular function for decorin in the aqueous humor outflow.

Primary open-angle glaucoma, a neurodegenerative disorder characterized by degeneration of optic nerve axons, is a frequent cause of vision loss and blindness worldwide. Several randomized multicenter studies have identified intraocular pressure as the major risk factor for its development, caused by an increased outflow resistance to the aqueous humor within the trabecular meshwork. However, the molecular mechanism for increased outflow resistance in POAG has not been fully established. One of the proposed players is the pro-fibrotic transforming growth factor (TGF)-ß2, which is found in higher amounts in the aqueous humor of patients with POAG. In this study we elucidated the role of decorin, a small leucine-rich proteoglycan and known antagonist of TGF-ß, in the region of aqueous humor outflow tissue. Utilizing decorin deficient mice, we discovered that decorin modulated TGF-ß signaling in the canonical outflow pathways and the lack of decorin in vivo caused an increase in intraocular pressure. Additionally, the Dcn-/- mice showed significant loss of optic nerve axons and morphological changes in the glial lamina, typical features of glaucoma. Moreover, using human trabecular meshwork cells we discovered that soluble decorin attenuated TGF-ß2 mediated synthesis and expression of typical downstream target genes including CCN2/CTGF, FN and COL IV.  Finally, we found a negative reciprocal regulation of decorin and TGF-ß, with a dramatic downregulation of decorin in the canonical outflow pathways of patients with primary open-angle glaucoma. Collectively, our results indicate that decorin plays an important role in the pathogenesis of primary open-angle glaucoma and offers novel perspectives in the treatment of this serious disease.

CCN2/CTGF promotor activity in the developing and adult mouse eye.

CCN2/CTGF is a matricellular protein that is known to enhance transforming growth factor-ß signaling and to induce a myofibroblast-like phenotype in a variety of cell types. Here, we investigated Ccn2/Ctgf promotor activity during development and in the adult mouse eye, using CTGFLacZ/+ mice in which the ß-galactosidase reporter gene LacZ had been inserted into the open reading frame of Ccn2/Ctgf. Promotor activity was assessed by staining for ß-galactosidase activity and by immunolabeling using antibodies against ß-galactosidase. Co-immunostaining using antibodies against glutamine synthetase, glial fibrillary acidic protein, choline acetyltransferase, and CD31 was applied to identify specific cell types. Ccn2/Ctgf promotor activity was intense in neural crest-derived cells differentiating to corneal stroma and endothelium, and to the stroma of choroid, iris, ciliary body, and the trabecular meshwork during development. In the adult eye, a persistent and very strong promotor activity was present in the trabecular meshwork outflow pathways. In addition, endothelial cells of Schlemm's canal, and of retinal and choroidal vessels, retinal astrocytes, Müller glia, and starburst amacrine cells were stained. Very strong promoter activity was seen in the astrocytes of the glial lamina at the optic nerve head. We conclude that CCN2/CTGF signaling is involved in the processes that govern neural crest morphogenesis during ocular development. In the adult eye, CCN2/CTGF likely plays an important role for the trabecular meshwork outflow pathways and the glial lamina of the optic nerve head.

Angiopoietin-1 Mimetic Nanoparticles for Restoring the Function of Endothelial Cells as Potential Therapeutic for Glaucoma.

A root cause for the development and progression of primary open-angle glaucoma might be the loss of the Schlemm's canal (SC) cell function due to an impaired Angiopoietin-1 (Angpt-1)/Tie2 signaling. Current therapeutic options fail to restore the SC cell function. We propose Angpt-1 mimetic nanoparticles (NPs) that are intended to bind in a multivalent manner to the Tie2 receptor for successful receptor activation. To this end, an Angpt-1 mimetic peptide was coupled to a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block co-polymer. The modified polymer allowed for the fabrication of Angpt-1 mimetic NPs with a narrow size distribution (polydispersity index < 0.2) and the size of the NPs ranging from about 120 nm (100% ligand density) to about 100 nm (5% ligand density). NP interaction with endothelial cells (HUVECs, EA.hy926) as surrogate for SC cells and fibroblasts as control was investigated by flow cytometry and confocal microscopy. The NP-cell interaction strongly depended on the ligand density and size of NPs. The cellular response to the NPs was investigated by a Ca2+ mobilization assay as well as by a real-time RT-PCR and Western blot analysis of endothelial nitric oxide synthase (eNOS). NPs with a ligand density of 25% opposed VEGF-induced Ca2+ influx in HUVECs significantly which could possibly increase cell relaxation and thus aqueous humor drainage, whereas the expression and synthesis of eNOS was not significantly altered. Therefore, we suggest Angpt-1 mimetic NPs as a first step towards a causative therapy to recover the loss of SC cell function during glaucoma.

Egr2-guided histone H2B monoubiquitination is required for peripheral nervous system myelination.

Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.

Design of dye and superparamagnetic iron oxide nanoparticle loaded lipid nanocapsules with dual detectability in vitro and in vivo.

Lipid nanocapsules are treasured nanoparticulate systems, although they lack detectability in biological environments. To overcome this, we designed LNCs loaded simultaneously with fluorescent dye and superparamagnetic iron oxide nanoparticles (Dual LNCs). The introduction of both labels did not alter nanoparticle characteristics such as size (50 nm), size distribution (polydispersity index < 0.1) or surface modifications, including the effectiveness of targeting ligands. Furthermore, the colloidal stability, particle integrity and biocompatibility of the nanoparticles were not negatively affected by label incorporation. These Dual LNCs are concomitantly visualizable via fluorescence and transmitted light imaging after either the internalization by cells or systemic administration to mice. Importantly, they are detectable in liver sections of mice using transmission electron microscopy without additional enhancement. The iron content of 0.24% (m/m) is sufficiently high for precise quantification of nanoparticle concentrations via inductively coupled plasma optical emission spectroscopy. Dual LNCs are precious tools for the investigation of in vitro and in vivo performances of lipid nanocapsule formulations, since they allow for the use of complementary imaging methods for broad range detectability.

Endogenous Wnt/ß-catenin signaling in Müller cells protects retinal ganglion cells from excitotoxic damage.

Purpose: To analyze whether activation of endogenous wingless (Wnt)/ß-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage. Methods: Transgenic mice with a tamoxifen-dependent ß-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal ß-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed. Results: Following NMDA injection of wild-type mice, a statistically significant increase in retinal ß-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific ß-catenin deficiency. Furthermore, in mice with a ß-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a ß-catenin deficiency in Müller cells. Conclusions: Endogenous Wnt/ß-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.

Norrin Protects Retinal Ganglion Cells from Excitotoxic Damage via the Induction of Leukemia Inhibitory Factor.

PURPOSE: To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is a secreted protein that protects RGC from N-methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2. METHODS: Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment. RESULTS: After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice. CONCLUSIONS: Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.