Neurotrophic effect of a novel TrkB agonist on retinal ganglion cells

Abstract

Purpose: Retinal ganglion cells (RGCs) die in glaucoma and virtually all optic neuropathies. Recently, novel tropomyosin-related kinase B (TrkB) monoclonal antibodies have been shown to activate TrkB receptors and exert neuroprotective and neurotrophic effects. In the present study, the authors examined the ability of one of them, 29D7, to elicit RGC survival and neurite growth both in culture and in vivo.

Methods: RGCs from postnatal day (P)3 to P4 Sprague-Dawley rats were isolated by sequential immunopanning using a monoclonal antibody to Thy1. RGCs were cultured in serum-free defined medium in 96-well plates. RGC viability was assessed after 1 to 3 days by MTT assay. Activation of TrkB and downstream signaling molecules was confirmed by Western blot analysis. Intravitreal injections of 29D7 were performed after optic nerve axotomy, and subsequent RGC survival was quantified using beta-III tubulin immunostaining. Regeneration was assessed using retrograde fluorogold tracing in an optic nerve-peripheral nerve graft model.

Results: Similar to brain-derived neurotrophic factor (BDNF), the 29D7 antibody strongly promoted RGC survival and neurite growth in vitro compared with medium alone or control IgG. Forskolin, which weakly supported RGC survival on its own, potentiated the effect of 29D7. Intravitreal injection of 29D7 enhanced RGC survival but not regeneration in vivo 2 weeks after optic nerve injury.

Conclusions: Together, these findings demonstrate the potential for antibody-mediated TrkB agonism as a potential therapeutic approach to enhance RGC survival after optic nerve injury. Further studies are needed to elucidate the mechanistic differences between this TrkB agonist and BDNF.

Figure 1.

Effect of 29D7, BDNF, and forskolin on RGC survival in vitro. RGCs were purified from postnatal day 3 retinas and cultured in NB medium alone, with control IgG (10 μg/mL), 29D7 (10 μg/mL), or BDNF (50 ng/mL), alone or combined with forskolin (5 nM) as marked. Survival was assessed after 1, 2, and 3 days in vitro by MTT assay (results are mean ± SEM, N = 3; results of a representative experiment are shown; all experiments were repeated at least three times with similar results).

Figure 2.

Dose-response curves of RGC survival at 1 day in response to (A) 29D7 or to (B) 29D7 or BDNF in the presence of a cAMP analogue, CPT-cAMP (50 μM). Details of assay are the same as in Figure 1.

Figure 3.

Effects of 29D7, BDNF, and forskolin on RGC neurite growth. Approximately 2000 purified P3 RGCs were plated onto PDL-coated 96 wells for 1 to 3 days in vitro, as indicated. RGCs were labeled with calcein (1 mg/mL) for 1 hour. Three pictures were randomly taken in each well. The percentage of surviving RGCs, surviving RGCs with neurites longer than one cell body, and average neurite length per surviving cell cultured in the indicated factors were assessed (results are mean ± SEM; n > 200; N = 3; results of a representative experiment are shown; all experiments were repeated at least three times).

Figure 4.

Effect of control IgG, 29D7, and BDNF on intracellular signaling pathways in RGCs. Purified P3 RGCs (∼3 × 10⁵) were incubated in NB/0.02% BSA alone (NB control) or in NB containing 10 μg/mL control IgG, 29D7 antibody, or 50 ng/mL BDNF at 37°C for 2 hours. Protein lysates were immunoblotted for (A) phospho-Trk, (B) phospho-Akt, or (C) phospho-ERK1/2. Membranes were stripped and reblotted for total TrkB, Akt, and ERK1/2. Band densities were calculated with graphics editing software, ratioed to total TrkB, Akt, or ERK1/2, and normalized to NB control (dashed line set as 100%). (*P < 0.05; N = 5 experiments, mean ± SEM shown).

Figure 5.

Retinal penetration and activity of 29D7 after intravitreal injection. Immunofluorescence images of retinal cryosections. 29D7 or mouse control IgG (10 μg in 3 μL) was injected into the vitreous of adult rats. At 1 and 2 days later, rats were euthanatized, and immunostaining against mouse IgG was performed. A peak in immunofluorescence was seen at 48 hours for both the control antibody and 29D7. Insets: colocalization of FG and immunostaining. Scale bar, 50 μm (125 μm in insets).

Figure 6.

Effect of 29D7 or BDNF on adult RGC survival at 14 days after optic nerve (ON) injury in vivo. The average number of surviving βIII tubulin–positive RGCs in retinas after complete ON transection and (A) one or (B) two intravitreal injections. (A) In animals receiving a single injection at day 3, no significant differences were found between control IgG and 29D7 groups (P = 0.15, unpaired t-test with Welch correction). There was a significant protective effect with BDNF (*P < 0.05; one-way ANOVA with Bonferroni's posttest). (B) In animals receiving two injections, at days 3 and 10, 29D7 and BDNF significantly enhanced RGC survival (***P < 0.001, one-way ANOVA with Bonferroni's test, 29D7 or BDNF compared with the other groups, and BDNF vs. 29D7; n = 5–7 animals per group; mean ± SEM shown).