Abstract
Purpose :
Since retinal ganglion cell (RGC) axons cannot spontaneously regenerate after injury, there is an unmet need to identify molecular regulators for RGC axon regeneration. In this study, we investigate the role of the Tubulin Polymerization Promoting Protein family member 3 (Tppp3) in axon regeneration using an optic nerve crush (ONC) model in the rodent.
Methods :
AAV2-Tppp3 overexpression and AAV2-Tppp3 shRNA virus were validated in ARPE 19 cells and probed by Western blot. Primary RGCs isolated from P3 mice pups by immunopanning were used to study neurite outgrowth. Primary RGCs were transduced with AAV2-Tppp3 overexpression or AAV2-control and AAV2-Tppp3 shRNA or AAV2-scramble for three days. Primary RGCs were immunostained with Tuj-1 to visualize neurite outgrowth and quantified using ImageJ. 8-10 week-old mice were treated with AAV2-Tppp3 overexpression or AAV2-control by intravitreal injection in OS two weeks before ONC. Animals were sacrificed for RGC survival, and axon regeneration studies two weeks after ONC. Flatmount retinas were stained with RBPMS for RGC counting. Regenerative axons were visualized by CTB-555. RNA sequencing was performed on mice retinas that were collected two days after ONC. The study was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by IACUC at the University of Pittsburgh. All experiments were performed in at least independent triplicate, analyzed by Student’s t-test or one-way ANOVA, and considered significant if P < 0.05.
Results :
Tppp3 protein was elevated and reduced after adding AAV2-Tppp3 overexpression and AAV2-Tppp3 shRNA viruses. Primary RGCs transduced with AAV2-Tppp3 overexpression virus showed a significant increase in neurite outgrowth compared with AAV2-control. However, downregulation of Tppp3 by AAV2-Tppp3 shRNA did not affect RGC neurite outgrowth. In-vivo, we observed that AAV2-Tppp3 overexpression promoted axon regeneration two weeks after ONC and showed improved RGC survival. Further RNA sequencing results reveal that various genes, including inflammation-related markers, are differentially expressed two days after ONC after Tppp3 treatment.
Conclusions :
In this study, we identified the role of Tppp3 in promoting RGC survival and axon regeneration. Tppp3 acts as a regulator to stimulate the intrinsic regenerative ability of RGCs, which would provide a translational impact on regenerative medicine.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.