Nogo-A-targeting antibody promotes visual recovery and inhibits neuroinflammation after retinal injury

Abstract

N-Methyl-D-aspartate (NMDA)-induced neuronal cell death is involved in a large spectrum of diseases affecting the brain and the retina such as Alzheimer's disease and diabetic retinopathy. Associated neurological impairments may result from the inhibition of neuronal plasticity by Nogo-A. The objective of the current study was to determine the contribution of Nogo-A to NMDA excitotoxicity in the mouse retina. We observed that Nogo-A is upregulated in the mouse vitreous during NMDA-induced inflammation. Intraocular injection of a function-blocking antibody specific to Nogo-A (11C7) was carried out 2 days after NMDA-induced injury. This treatment significantly enhanced visual function recovery in injured animals. Strikingly, the expression of potent pro-inflammatory molecules was downregulated by 11C7, among which TNFα was the most durably decreased cytokine in microglia/macrophages. Additional analyses suggest that TNFα downregulation may stem from cofilin inactivation in microglia/macrophages. 11C7 also limited gliosis presumably via P.Stat3 downregulation. Diabetic retinopathy was associated with increased levels of Nogo-A in the eyes of donors. In summary, our results reveal that Nogo-A-targeting antibody can stimulate visual recovery after retinal injury and that Nogo-A is a potent modulator of excitotoxicity-induced neuroinflammation. These data may be used to design treatments against inflammatory eye diseases.

Fig. 1. Retinal injury induces Nogo-A protein release in the mouse vitreous.

a–c Compared with intact condition (no treatment) and PBS treatment, intravitreal injection of NMDA induced the loss of retinal ganglion cells expressing Nogo-A and the specific cell marker RBPMS. In contrast, glial expression of Nogo-A did not change in glutamine synthetase (GS)-expressing Müller cells after NMDA-induced injury. Three mice per condition were examined. d By Western Blotting, the level of Nogo-A protein did not significantly change in retinal lysates treated with increasing doses of NMDA (0–40 nmoles) although P.Stat₃ and P.Erk_1/2 were upregulated in a dose-dependent manner in response to retinal damage. e Western Blot analysis of mouse vitreous (4 μL/well) revealed that NMDA induced the increase of Nogo-A and TNFα proteins in a dose-dependent manner. For quantitative analysis, 3 mice were used per condition. With Rb173A antibody which recognizes the Nogo-A specific domain encoded by exon 3, 4 proteins were observed at ~80 kDa (Nogo-A-F1), ~68 kDa (Nogo-A-F2), and at ~200–250 kDa (Full-length Nogo-A, Nogo-A-FL). Strikingly, TNFα appeared predominantly in trimers, i.e. under its most inflammatory form. f, g Additional antibodies directed against different parts of Nogo-A, named Rb1, S544, and 11C7, were used to determine the presence of Nogo-A proteins in the vitreous of NMDA-treated eyes. Increased levels of Nogo-A proteins were thus confirmed. Interestingly, proteins of similar molecular weight were found and may correspond to Nogo-A-F1, Nogo-A-F2, and Nogo-A-FL. Statistics: One-way ANOVA, Dunnett’s post hoc test, *P < 0.05; **P < 0.01; ***P < 0.001; ^†P < 0.0001. Scale bars: a, b close-up (bottom left) = 25 μm; b (top left) = 100 μm.

Fig. 2. Intravitreal administration of Nogo-A blocking antibody enhances visual recovery after retinal injury.

a To follow visual function changes following NMDA-induced injury (1), the optokinetic response was monitored before and after intravitreal injections (2). Control IgG (2μg/eye) or anti-Nogo-A IgG (11C7, 2 μg/eye) was delivered in the vitreous 2 days after NMDA injection. Electroretinograms were recorded in photopic conditions 6 weeks after NMDA injections (3, see Fig. S1). Retinal ganglion cell survival was assessed on retinal flat-mounts stained for RBPMS, the day following ERG recordings (4). At 0.5 nmol of NMDA, nine mice were examined for each antibody treatment (control IgG or 11C7). Six mice received control IgG and 11C7 after the injection of 2 nmol NMDA. b Immunofluorescent staining with RBPMS antibody revealed a ~30% reduction in the density of RGCs after injection of 0.5 nmol of NMDA and a ~70-% reduction following the administration of 2 nmol of NMDA relative to intact retinae. The level of cell death did not statistically vary between the two groups receiving either 11C7 or control IgG. c The optokinetic response of mice receiving 0.5 nmol of NMDA showed significant function deficits after control IgG treatment. Similar visual loss was obtained with 0.5 nmol NMDA alone, without antibody (data not shown). In contrast, blocking Nogo-A with 11C7 allowed much better recovery of optokinetic response sensitivity. The effect of 11C7 was not significant when injected after 2 nmol of NMDA. d The optokinetic response of individual animals revealed weak variability in groups injected with 0.5 nmol of NMDA compared with 2 nmol of NMDA. Statistics: c, ***P < 0.001, two-way ANOVA; d, ***P < 0.001, unpaired t-test. Scale bar = 100 μm.

Fig. 3. Antibody-based neutralization of Nogo-A mitigates inflammation in the injured retina.

a, b Retinal gene expression analysis by qRT-PCR at 3 and 7 days after NMDA injection (0.5 nmol) and antibody treatments (2 μg/eye). Four-six mice were used per condition. 11C7 reduced the expression of major genes involved in inflammation and monocyte activation (*P < 0.05; **P < 0.01; unpaired t-test). In particular, 11C7 injection led to sustained decrease of Tnf transcript and Sprr1a mRNA, the expression of which reflects the severity of neuronal injury response. c The level of TNFα trimers was studied by Western Blotting the day after antibody injection. Compared with control IgG (n = 3 mice), 11C7 injections (n = 4 mice) significantly decreased the content of TNFα in the vitreous of injured eyes (**P < 0.01, Unpaired t-test). d TNFα was mainly observed in Iba1-expressing monocytes by immunofluorescence on retinal flat-mounts. Its signal was much weaker with 11C7 than in control mice. Immunofluorescent staining observations were repeated in three different mice for each antibody treatment. Scale bar in d = 100 μm.

Fig. 4. Antibody-mediated Nogo-A blockade decreases the number of cells expressing TNFα without affecting the number of Iba1-positive monocytes.

Cells expressing TNFα, Iba1, and CD68 were labeled on cryosections by immunofluorescence in 4 mouse retinae, 24 h after control IgG or 11C7 injection. a Retinal sections showed many TNFα-containing cells located in the inner retina, between the outer plexiform layer and the ganglion cell layer 1 day after control IgG injection. A majority of cells was positive for Iba1 and exhibited vesicular staining of the lysosomal protein CD68, indicating their active state. b The injection of 11C7 strongly attenuated TNFα and CD68 expression and to a lower extent the intensity of Iba1 in macrophages/microglia. c Quantification of the number of cells expressing TNFα and Iba1 suggesting potent and specific effects of 11C7 on TNFα cytokine reduction. Six sections were examined for each retina. Statistics: one-way ANOVA, Dunnett’s post hoc test, **P < 0.01; ****P < 0.0001. Scale bars = 50 μm.

Fig. 5. 11C7 treatment inhibits inflammatory processes in injured retinal glia.

a, b Western blot analysis of whole retina lysates allowed to detect marked changes in the phosphorylation level of cofilin, P.Stat3 and P.Erk1/2 after NMDA/11C7 injection (n = 5 mice) compared with control eye treated with NMDA/control IgG (n = 4 mice). In its phosphorylated state, cofilin is inactivated by 11C7. In parallel, P.Stat3 downregulation suggests cytokine signaling reduction. c By immunofluorescence on retinal cross sections, P.cofilin upregulation was visualized in microglia/macrophages labeled with Iba1 (arrows). d P.Stat3 decrease was from Müller glia positive for glutamine synthetase (GS) and from isolectin B4 (IB4)-labeled blood vessels. e Following NMDA and 11C7 treatments, P.Erk1/2 immunoreactivity increased in the nucleus and in the endfeet (EF) of Müller cells identified with cytoplasmic GS. Immunofluorescent stainings were repeated in 3 mice/condition. Scales bars: c–e = 100 μm, close-up in d = 25 μm. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Statistics: *P < 0.05; **P < 0.01; Unpaired t-test.

Fig. 6. Nogo-A protein is increased in the vitreous and in the retina of patients affected by diabetic retinopathy.

a Western blot analysis was carried out to detect Nogo-A protein in the vitreous of donors. Anti-Nogo-A antibody allowed to detect recombinant human Nogo-A protein. b In the vitreous of non-diabetic patients, Nogo-A signal was very weak in comparison to patients with diabetes and proliferative diabetic retinopathy. c Immunofluorescent stainings in human retinal sections allowed to visualize Nogo-A in cell bodies (arrows) of the ganglion cell layer (GCL) and in Müller cell endfeet (EF). Dapi was used to recognize retinal cell layers in blue. To distinguish astrocytes from Müller cell processes in the nerve fiber layer (NFL), GFAP and GS were used respectively as specific cell markers. Without diabetes, astrocytes are the only glia expressing GFAP. They were deprived of Nogo-A staining. In contrast, Nogo-A was colocalized with GS in Müller cell endfeet. d In the retina of a diabetic patient, Nogo-A and GS were upregulated in Müller cells whose radial processes span the whole retinal thickness. e In the macula of a diabetic retinopathy (DR) patient, severe cystoid macular edema (CME) was observed. Around prominent cysts, Nogo-A and GFAP expression was strongly upregulated in gliotic Müller cells. These observations link Nogo-A expression increase with different stages of diabetic retinopathy. Scale bars = 20 μm.