A potential therapeutic target for optic nerve regeneration

Abstract

Purpose : Retinal ganglion cell (RGC) axons cannot spontaneously regenerate after injury in mammals. There is an unmet need to identify therapeutic targets for RGC axon regeneration. In this study, we investigate the function of the Tubulin Polymerization Promoting Protein family member 3 (Tppp3) in RGC survival and axon regeneration using an optic nerve crush (ONC) rodent model.

Methods : Primary mouse RGCs were isolated from P3 pups by immunopanning. RGCs were transduced with AAVs either overexpressing Tppp3 or knocking down Tppp3 or control vectors for neurite outgrowth observation, detecting by Tuj-1 staining. E14.5 retinas were dissociated and treated with mouse GDF15, GDF11, or PBS for 5 days for 10x genomic scRNA Sequencing. Open-source single cell RNA (scRNA) sequencing datasets were used for macaque and human samples. In vivo, 8-10 week-old mice were intravitreal injected with Tppp3 overexpression or control AAV two weeks before ONC. RGC survival and axon regeneration were assessed two weeks post-ONC using RBPMS staining for pan-RGCs, and Opn4 staining for ipRGCs. CTB-555 was used to visualize regenerative axons. Bulk-RNA sequencing was performed on mice retinas collected two days post-ONC. The study was conducted in compliance with the ARVO guidelines and approved by IACUC at the University of Pittsburgh and Stanford. Experiments were conducted in triplicate and analyzed with Student’s t-test or one-way ANOVA, with significance set at P < 0.05.

Results : In immunostaining, in situ hybridization, Western blot, and scRNA seq data, we found that Tppp3 is highly expressed in the RGCs among mice. scRNA seq data in macaque and human retinas also shows Tppp3 within RGC clusters. Tppp3 expression is also downregulated by an RGC fate suppressor, suggesting Tppp3 may be involved in RGC differentiation. In vitro data showed that Tppp3 positively regulates mouse RGC neurite outgrowth. In vivo data further showed that Tppp3 overexpression promotes axon regeneration and RGC survival by upregulating inflammation-related genes and possibly through the BMP4 signaling. Interestingly, Tppp3 treatment showed a trend in preserving Opn4+ RGCs after ONC.

Conclusions : In this study, we identified Tppp3 as an RGC marker and revealed its function in RGC survival and axon regeneration after optic nerve injury. The synergistic potential with other axon regeneration regulators highlights Tppp3 as a possible therapeutic target in CNS regenerative medicine.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.