A Small Molecule TrkB Neurotrophin Receptor Partial Agonist as Possible Treatment for Experimental Nonarteritic Anterior Ischemic Optic Neuropathy

Abstract

Purpose: Brain-derived neurotrophic factor (BDNF) and activation of its high affinity receptor tropomyosin kinase (Trk) B promote retinal ganglion cells (RGCs) survival following injury. In this study, we tested the effects of LM22A-4, a small molecule TrkB receptor-specific partial agonist, on RGC survival in vitro and in experimental nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in those older than 50 years.

Methods: We assessed drug effects on immunopanned, cultured RGCs and calculated RGC survival and assessed TrkB receptor activation by mitogen-activated protein (MAP) kinase translocation. To assess effects in vivo, we induced murine AION and treated the animals with one intravitreal injection and three-week systemic treatment. We measured drug effects using serial spectral-domain optical coherence tomography (OCT) and quantified retinal Brn3A⁺ RGC density three weeks after ischemia.

Results: In vitro, LM22A-4 significantly increased the survival of cultured RGCs at day 2 (95% CI control: 8.4-13.6; LM22A-4: 23.7-30.3; BDNF: 24.3-29.9; P ≤ 0.0001), similar to the effect of the endogenous TrkB receptor ligand BDNF. There was also significant nuclear and cytoplasmic translocation of MAP kinase (95% CI control: 0.9-6.8; LM22A-4: 38.8-84.4; BDNF: 64.0-93.0; P = 0.0002), a known downstream event of TrkB receptor activation. Following AION, LM22A-4 treatment led to significant preservation of the ganglion cell complex (95% CI: AION-PBS: 66.8-70.7%; AION-LM22A-4: 70.0-73.1; P = 0.03) and total retinal thickness (95% CI: AION-PBS: 185-196%; AION-LM22A-4: 195-203; P = 0.002) as measured by OCT compared with non-treated eyes. There was also significant rescue of the Brn3A⁺ RGC density on morphometric analysis of whole mount retinae (95% CI control: 2360-2629; AION-PBS: 1647-2008 cells/mm²; AION-LM22A-4: 1958-2216 cells/mm²; P = 0.02).

Conclusions: TrkB receptor partial agonist LM22A-4 promoted survival of cultured RGCs in vitro by TrkB receptor activation, and treatment in vivo led to increased survival of RGCs after optic nerve ischemia, providing support that LM22A-4 may be effective therapy to treat ischemic optic neuropathy.

Abbreviations: AION: anterior ischemic optic neuropathy, BDNF: Brain-derived neurotrophic factor, GCC: ganglion cell complex, MAP: mitogen-activated protein, OCT: spectral-domain optical coherence tomography, OD: right eye, ON: optic nerve, ONH: optic nerve head, OS: left eye, RGC: retinal ganglion cell; Trk: tropomyosin kinase.

Keywords: AION; BDNF; OCT; TrkB; optic neuropathy; pharmacophore; retinal ganglion cell.

Figure 1.

LM22A-4 treatment increased survival of cultured RGCs on day 2. (A) Purified RGCs cultured stained with β-tubulin antibody and DAPI. Many cells exhibited short processes in all conditions. (B) Live-dead assay of RGCs using calcein-AM and ethidium bromide to calculate survival. (C) Bar graph of survival assay that showed treatment with LM22A-4 significantly increased RGC survival (P < 0.0001) similar to the effect of BDNF.

Figure 2.

LM22A-4 treatment promoted TrkB receptor activation and MAP kinase translocation in cultured RGCs on day 2. (A) Control. (B) LM22A-4 treated. (C) BDNF-treated. Top row: lower magnification images. Middle and bottom rows: higher magnification images. (D) Bar graph of MAP kinase distribution in a diffuse or punctate, pattern. In control condition in (A), the RGCs exhibited diffuse anti-MAP kinase staining in the soma and processes. In LM22A-4 and BDNF treated groups (B,C) there was a significant change in the distribution with punctate appearance of anti-MAP kinase staining in and around the nuclei and decreased and punctate distribution in the neurites.

Figure 3.

In vivo treatment with LM22A-4 after experimental AION led to preserved retinal thickness measured using SD-OCT. (A) Top: example of fundus photograph and B-scan using the circular scan pattern. Bottom: Bar graph of GCC measurements in different treatment groups showing there was significant preservation of GCC layer thickness in the AION group after three weeks of LM22A-4 treatment (N = 23, P = 0.03). (B) Top: example of fundus photograph and B-scan using the posterior pole analysis. TRT: total retinal thickness. Bottom: bar graph of total retinal thickness of the optic disc week-3 after AION showing LM22A-4 treatment significantly improved optic disc thickness (N = 46, P = 0.002). GCC: Ganglion cell layer

Figure 4.

In vivo LM22A-4 treatment led to increased survival of Brn3A⁺ RGCs after AION. (A) Representative images of Brn3A⁺ RGCs in PBS-treated and LM22A-4 treated groups at week-3. (B) Bar graph of quantification of Brn3A⁺ cells in control and AION eyes treated with LM22A-4 showing significant increase in Brn3A⁺ RGCs after LM22A-4 treatment in the AION groups (P = 0.02). (C) High correlation of the automatic and manual RGC counting methods