Roswitha Seitz

Norrin mediates neuroprotective effects on retinal ganglion cells via activation of the Wnt/beta-catenin signaling pathway and the induction of neuroprotective growth factors in Muller cells.

Norrin is a secreted protein that binds to frizzled 4 and controls development of capillaries in retina and inner ear. We provide evidence that Norrin has distinct neuroprotective properties that are independent from its effects on vascular development. The function of Norrin was investigated in a mouse model of excitotoxic retinal ganglion cell (RGC) damage after intravitreal injection of NMDA, and in cultured Müller glia or immortalized RGC-5 cells. Intravitreal injection of Norrin significantly increased the number of surviving RGC axons in the optic nerve and decreased apoptotic death of retinal neurons following NMDA-mediated damage. This effect could be blocked by adding dickkopf (DKK)-1, an inhibitor of the Wnt/β-catenin signaling pathway. Treatment of eyes with combined Norrin/NMDA activated Wnt/β-catenin signaling and increased the retinal expression of leukemia inhibitory factor and endothelin-2, as well as that of neurotrophic growth factors such as fibroblast growth factor-2, brain-derived neurotrophic factor, lens epithelium-derived growth factor, and ciliary neurotrophic factor. A similar activation of Wnt/β-catenin signaling and an increased expression of neurotrophic factors was observed in cultured Müller cells after treatment with Norrin, effects that again could be blocked by adding DKK-1. In addition, conditioned cell culture medium of Norrin-treated Müller cells increased survival of differentiated RGC-5 cells. We conclude that Norrin has pronounced neuroprotective properties on retinal neurons with the distinct potential to decrease the damaging effects of NMDA-induced RGC loss. The effects of Norrin involve activation of Wnt/β-catenin signaling and subsequent induction of neurotrophic growth factors in Müller cells.

Norrin Promotes Vascular Regrowth after Oxygen-Induced Retinal Vessel Loss and Suppresses Retinopathy in Mice

Norrin is a secreted protein that is involved in retinal angiogenesis and activates the Wnt-signaling pathway. We studied the role of Norrin in microvascular endothelial cells in vitro, and in a mouse model of retinopathy characterized by oxygen-induced vascular loss followed by hypoxia-induced pathological neovascularization. Recombinant Norrin significantly increased proliferation, viability, migration, and tube formation in vitro. Two independent transgenic mouse strains with ectopic overexpression of Norrin from the lens (βB1-Crystallin-Norrin), or the retinal pigment epithelium (Rpe65-Norrin) were generated and exposed to high oxygen. Following oxygen treatment, vascular loss was significantly smaller in retinae of transgenic mice from both strains as compared to wild-type littermates. In addition, the anatomical correct regrowth of vessels was significantly increased, while pathological neovascularization was suppressed. In vitro and in vivo effects of Norrin could be blocked by adding DKK (Dickkopf)-1, an inhibitor of Wnt/β-catenin signaling. Treatment of microvascular endothelial cells with Norrin caused a substantial increase in the expression of angiopoietin-2 (Ang-2). When inhibitory antibodies against Ang-2 were added to Norrin, the proliferative effects of Norrin were significantly suppressed. We conclude that Norrin is a potent factor to induce angiogenesis in microvascular endothelial cells, which has the distinct potential to suppress the damaging effects of oxygen-induced retinopathy in vivo. The effects of Norrin appear to be mediated, at least partially, via the induction of Ang-2.

The role of Müller glia and microglia in glaucoma.

Cells of Müller glia and microglia react to neuronal injury in glaucoma. The change to a reactive phenotype initiates signaling cascades that may serve a neuroprotective role, but may also proceed to promote damaging effects on retinal neurons. Both effects appear to occur most likely in parallel in glaucoma, but the underlying mechanisms and signaling pathways that specifically promote protective versus destructive roles of reactive glial cells are mostly unclear. More research is needed to understand the homeostatic signaling network in which retinal glia cells are embedded to maintain or restore neuronal function after injury.

N-methyl-D-aspartate (NMDA)-mediated excitotoxic damage: a mouse model of acute retinal ganglion cell damage.

The animal model of N-methyl-D-aspartate (NMDA)-induced excitotoxic damage of retinal ganglion cells (RGC) is widely used to study the molecular mechanisms of RGC apoptosis and/or its prevention by neuroprotective agents. This chapter provides protocols for applying NMDA-induced excitotoxic damage to RGC of mouse eyes and for subsequent measuring of the extent of the resulting damage.

Norrin protects optic nerve axons from degeneration in a mouse model of glaucoma.

Norrin is a secreted signaling molecule activating the Wnt/β-catenin pathway. Since Norrin protects retinal neurons from experimental acute injury, we were interested to learn if Norrin attenuates chronic damage of retinal ganglion cells (RGC) and their axons in a mouse model of glaucoma. Transgenic mice overexpressing Norrin in the retina (Pax6-Norrin) were generated and crossed with DBA/2J mice with hereditary glaucoma and optic nerve axonal degeneration. One-year old DBA/2J/Pax6-Norrin animals had significantly more surviving optic nerve axons than their DBA/2J littermates. The protective effect correlated with an increase in insulin-like growth factor (IGF)-1 mRNA and an enhanced Akt phosphorylation in DBA/2J/Pax6-Norrin mice. Both mouse strains developed an increase in intraocular pressure during the second half of the first year and marked degenerative changes in chamber angle, ciliary body and iris structure. The degenerations were slightly attenuated in the chamber angle of DBA/2J/Pax6-Norrin mice, which showed a β-catenin increase in the trabecular meshwork. We conclude that high levels of Norrin and the subsequent constitutive activation of Wnt/β-catenin signaling in RGC protect from glaucomatous axonal damage via IGF-1 causing increased activity of PI3K-Akt signaling. Our results identify components of a protective signaling network preventing degeneration of optic nerve axons in glaucoma.

Cross-Inhibition of Norrin and TGF-β Signaling Modulates Development of Retinal and Choroidal Vasculature

Purpose Norrin is essential for the formation of the retinal vasculature during development and promotes its repair after damage via activation of Wnt/β-catenin signaling. Since retinal TGF-β signaling has essentially opposite effects on the retinal vasculature we investigated if and how Norrin inhibits TGF-β signaling, and vice versa. Methods Eyes from transgenic mice with an overexpression of Norrin (βB1-Norrin) and/or active TGF-β (βB1-TGF-β1) in the lens were generated and analyzed by light microscopy, immunohistochemistry, and TUNEL. Further on, protein as well as mRNA levels were investigated by Western blot analyses and real-time RT-PCR, respectively. Results In βB1-TGF-β1 mice, the lack of retinal vascular development and choriocapillaris maintenance was rescued when transgenic Norrin was additionally overexpressed in the eye. In addition, retinal Wnt/β-catenin signaling and the levels of SMAD7, an inhibitor of the canonical TGF-β pathway, were substantially suppressed in retinae of βB1-TGF-β1 mice. In contrast, Norrin normalized Wnt/β-catenin signaling and SMAD7 levels in double transgenic mice. Moreover, in retinae of βB1-TGF-β1 mice, the amounts of phosphorylated SMAD3, a downstream mediator of TGF-β signaling, were increased compared to those of βB1-Norrin/βB1-TGF-β1 mice. In vitro, Norrin substantially reduced the TGF-β-mediated induction of target genes, an effect that was blocked by Dickkopf-1, a specific inhibitor of Wnt/β-catenin signaling. Conclusions High amounts of TGF-β in the eye cause a substantial reduction in the activity of Wnt/β-catenin signaling. This effect is inhibited in the presence of high amounts of Norrin, which further induce the expression of SMAD7 to inhibit TGF-β signaling.