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.


Supplemental Experimental Procedures
Generation of Pax6-Norrin mice Two fragments of the Pax6 α-enhancer fragment were PCR amplified from murine genomic DNA using the primer pairs 5′-CTGGGAATTACCCTGGCT-3′ and 5′-GCGCGCACGCGTGTGGAATA-3′ (fragment 1; 1258 bp) and 5'-GGTAAAATCATAGACGCGCTCCTTC-3' and 5'-TGGGCAGCCCAGCCTCAAA-3' (fragment 2; 749 bp), and cloned into the pDrive vector according to the manufacturer's instructions (Qiagen). The endogenous BssHII restriction site of both fragments was used to insert fragment 1 into the pDrive fragment 2 vector after KpnI-BssHII digest to obtain the Pax6 α-enhancer fragment (1642bp). The P0 minimal promoter fragment was amplified using the primer pairs 5'-GAACCTAAGGACAGGCTACG-3' and 5'-CATGAATTCGGCGCGAGGCTTG-3´ (990bp) and cloned into the Topo blunt vector (Invitrogen). To introduce restriction sides for SacII at the 5'-end, an additional PCR with the primer pair 5'-CCGCGGGAACCTAAGGACAGGCTACG-3' and 5'-CATGAATTCGGCGCGAGGCTTG-3' (containing an endogenous EcoRI restriction site) was performed and the obtained SacII-P0 minimal promoter-EcoRI fragment was cloned into a Topo blunt vector. Following this, an additional PCR of the Pax6 αenhancer promoter fragment was performed using the primer pairs 5′-GAATTCGCCCTGCGGCCGCTGGGAATTACCCTGGCT-3′ and 5′-CCGCGGTGGGCAGCCCAGCCTCAAA-3′ to introduce at the 5′-end an EcoRI-NotI and at the 3′-end a SacII restriction site, and subcloned into the pDrive vector. After restriction digest with KpnI and SacII the EcoRI-NotI-Pax6 α-enhancer-SacII promoter fragment was cloned into the SacII-P0 minimal promoter-EcoRI Topo blunt plasmid to obtain the Pax6 α-enhancer-P0 promoter fragment, hereinafter referred as Pax6 promoter. All PCR products were sequenced before further cloning. The βB1-Crystallin promoter fragment of the βB1-Norrin plasmid 10 was replaced with the EcoRI-NotI-Pax6-EcoRI promoter fragment by EcoRI digest to obtain the plasmid Pax6-Norrin. Before microinjection, the construct was finally sequenced and released from the plasmid Pax6-Norrin by digest with NotI. For generation of transgenic mice, ES cells derived from FVB/N blastocysts were coelectroporated with the Pax6-Norrin expression construct and a circular neomycin resistance plasmid (PGK-Neo-bpA).

Northern blot analysis
For Northern blot analysis PCR was used to amplify a cDNA fragment of murine Norrin from plasmid Pax6-Norrin by using the primer pairs 5′-AGCTCAAAGATGGTGCTCCT-3′ and 5′-TAGAGCCAACAGGGGAAATG-3′ (product length, 495 bp). PCR products were gel-purified by using the Qiagen Gel purification kit (Qiagen) and cloned into pCR Topo TA vector (Invitrogen). After linearization of the vector with HindIII, an antisense RNA probe for Norrin was generated and labeled with DIG-11-UTP using T7-polymerase (Roche). For Northern blot analysis, 10 µg total RNA was separated on a 1% agarose gel containing 6 % formaldehyde and blotted onto a positively charged nylon membrane (Roche). After transfer, the blot was cross-linked using a UV Stratalinker 1800 (Stratagene). Prehybridization was performed for 1 h at 60°C using the Dig EasyHyb-buffer (Roche). After overnight hybridization at 60°C, membranes were washed for 5 min with 2x SSC and 0.1% SDS at room temperature and 15 min with 0.2x SSC and 0.1% SDS at 70°C. For detection of hybridization signals, membranes were blocked for 30 min at room temperature in 1% blocking reagent, 0.1 M maleic acid, and 0.15 M NaCl (pH 7.5) and incubated 30 min in anti-digoxigenin-alkaline phosphatase (1:10,000; Roche).
After washing membranes two times for 15 min in 0.1 m maleic acid, 0.15 m NaCl (pH 7.5) and 0.3% Tween 20, chemiluminescence detection was performed (CDP-Star; Roche). The membranes were visualized on a BAS 3000 Imager work station (Fujifilm). To monitor the integrity of RNA, the relative amounts of RNA loaded on the gel and the efficiency of transfer, membranes were stained with methylene blue. The intensity of the hybridization signal was determined by the Aida Image Analyzer v.4.06 software (Raytest).

Histological grading of the anterior eye segment
For semiquantitative analysis of the pathological changes, we used a grading system published previously that identifies no or mild, moderate and severe changes 21 .
Changes in chamber angle structure are no or mild when anterior synechiae cover only part of the trabecular meshwork, moderate when they completely cover trabecular meshwork and the very periphery of the cornea, and severe when synechiae extend more centrally onto the cornea (Fig. 6A). Changes in ciliary body structure are regarded as mild when the processes are shortened but epithelium and vascular structures appear normal, moderate when processes are atrophic with less distinct vascular and epithelial layers, and severe when process are not recognizable but atrophied to a flat epithelium (Fig. 6C). Iris changes are no or mild when the stroma is of normal thickness with fewer than normal iris pigment epithelium cells present, moderate when the stroma is thinner than normal and few or no pigment epithelium cells are present, and severe when the stroma is very thin and no pigment epithelium present (Fig. 6E).

Electrophysiology
Mice were dark adapted for at least 12 hours. Mice were anesthetized by subcutaneous injection of ketamine (65 mg/kg) and xylazine (13 mg/kg), and their pupils were dilated with tropicamide eyedrops (Mydriaticum Stulln). Silver needle electrodes served as reference (fore-head) and ground (tail) and gold wire ring electrodes as active electrodes. Corneregel (Bausch & Lomb) was applied to keep the eye hydrated and to maintain good electrical contact. ERGs were recorded using a Ganzfeld bowl (Ganzfeld QC450 SCX, Roland Consult) from both eyes simultaneously, band-pass filtered (1 to 300 Hz) and averaged. Single flash scotopic (dark adapted) responses to a series of ten LED-flash intensities ranging from -3.5 to 1 log cds/m 2 with an inter stimulus interval of 2 up to 20 s for the highest intensity were recorded. After 10 minutes of adaptation to a white background illumination (20 cd/m 2 ) single flash photopic (light adapted) responses to three Xenon-flash intensities (1, 1.5 and 2 log cds/m 2 ) were recorded. All analyses and plotting was carried out with R 3.3.2 and ggplot 2.2.1.