Abstract
Purpose :
Central Nervous System regeneration in mammals remains a challenge. POU class 2 homeobox 2 (Pou2f2), a gene expressed in retinal ganglion cells (RGCs), is involved in bone fracture healing and neuronal differentiation. Given that, we aim to understand whether overexpression of Pou2f2 can promote RGC survival and axon regeneration in an optic nerve crush (ONC) model.
Methods :
Pou2f2 was characterized by immunostaining of adult mouse retinal cryosection. Pou2f2 was encoded in a CMV-driven plasmid with a GFP tag and packed in AAV2. The protein expression efficiency was detected by Western blot. Overexpression (OE-Pou2h2), shRNA knockdown AAVs (shPou2f2), and their control AAVs were added to primary mouse RGCs from postnatal day 2 (P2) mice to observe the neurite outgrowth. AAVs were injected into C57/Bl6 mouse eyes 2 weeks before optic nerve crush (ONC), and CTB-555 was intravitreally injected into eyes 2 days before collection to observe the regenerative axons. The RGC survival would be determined by RBPMS staining of flat-mount retinas. The retinas from two weeks and two days after crush were collected for RNA sequencing. All animal procedures were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by IACUC at the University of Pittsburgh. All data were performed in at least independent triplicate, analyzed by Student’s t-test or by one-way ANOVA and considered significant if P < 0.05.
Results :
AAV virus infection efficiency was validated in ARPE-19 cells. Pou2f2 was highly expressed in the RGC layer. Overexpression of Pou2f2 promotes neurite length of RGC in-vitro, and knockdown of the Pou2f2 suppresses the neurite outgrowth. In vivo, Pou2f2 promotes RGC survival and axon regeneration in the ONC model. RNA-seq analysis revealed that the gene related to DNA repair was upregulated by OE-pou2f2 in the retina.
Conclusions :
This study showed that Pou2f2 is the potential promoting molecule for RGC regeneration. Overexpression of Pou2f2 via AAV infection could be a therapeutic strategy for treating RGC loss and axon degeneration in glaucoma and other optic neuropathies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.