Neuroprotection and axon regeneration in glaucoma models by gene therapy using modified tyrosine kinase receptors

Abstract

Aims/Purpose: To determine whether gene therapy using the modified tyrosine kinase receptors (insulin receptor or TrkB) can protect retinal ganglion cells (RGCs) and stimulate axon regeneration in glaucoma model mice.Methods: To examine the effects on the signalling pathways, Cos7 cells were transfected with intracellular region of insulin receptor (iIR) or TrkB (iTrkB) for 24 h, and immunoblot analysis was performed using antibodies against total and phosphorylated ERK and AKT. To evaluate their neuroprotective effects in vivo, the modified IR or TrkB was overexpressed in RGCs in GLAST knockout mice, a mouse model of normal tension glaucoma, by an intraocular injection of AAV‐iIR or AAV‐iTrkB. For optic nerve crush (ONC) model, AAV‐iIR or AAV‐iTrkB was injected into the mouse eyes 2 weeks before ONC. The number of surviving RGCs was examined by whole‐mount immunostaining of the retina with an RBPMS antibody. RGC dendrites were visualized by GFP labeling and quantified using the microscopy image analysis software IMARIS. Optic nerve regeneration was examined by the intraocular injection of Cholera Toxin Subunit B at 2 days before sacrifice.Results: iIR and iTrkB transfection in vitro both strongly induced phosphorylation of ERK and AKT, and in vivo, AAV‐iIR and AAV‐iTrkB both significantly increased the number of surviving RGCs in GLAST knockout mice and ONC mice. Moreover, AAV‐iIR and AAV‐iTrkB promoted axon regeneration in ONC mice and preserved the structure of RGC dendrites.Conclusions: Our system overcomes the small size limitation of the genome packaging in AAV and may be useful for the treatment of retinal and optic nerve degeneration.References Nishijima E, Honda S, Kitamura Y, Namekata K, Kimura A, Guo X, Azuchi Y, Harada C, Murakami A, Matsuda A, Nakano T, Parada LF, Harada T. Vision protection and robust axon regeneration in glaucoma models by membrane‐associated Trk receptors. Molecular Therapy 31(3), 810–824, 2023. doi:10.1016/j.ymthe.2022.11.018