Pou2f2 enhances retinal ganglion cell survival and axon regeneration after optic nerve injury

Retinal ganglion cell (RGC) degeneration and the limited regenerative capacity of mammalian central nervous system axons result in irreversible vision loss in optic neuropathies. Identifying intrinsic regulators that promote both neuroprotection and axon regeneration is critical for developing effective therapies. This study identified POU domain class 2 transcription factor 2 (Pou2f2) as a previously unrecognized regulator involved in RGC biology.

Single-cell RNA sequencing analysis of developing mouse and human retinas showed that Pou2f2 is enriched in RGC clusters and suppressed by an RGC fate suppressor in the mouse retinal progenitor cell culture. Pou2f2 protein was detected in mouse RGCs and optic nerves, where its expression decreased following optic nerve crush (ONC). In primary RGC cultures, AAV2-mediated Pou2f2 overexpression enhanced neurite outgrowth, whereas Pou2f2 knockdown reduced neurite length.

In vivo, intravitreal injection of Pou2f2 overexpression AAV2s increased axon regeneration, improved RGC survival, and partially preserved RGC function after ONC. Bulk RNA sequencing data revealed early activation of cytoskeletal growth pathways and later upregulation of DNA repair-associated genes, whereas immunostaining and in vitro analyses suggested activation of AKT/mTOR signaling following Pou2f2 overexpression. Together, these findings identify Pou2f2 as a promising molecular target for gene-based therapies for RGC degenerative diseases, including glaucoma and other optic neuropathies.

The authors are grateful to Dr. Larry Benowitz for the comments and scientific discussion. We thank the University of Pittsburgh Ophthalmology Core Facilities, including Dr.

Paul Kinchington (Molecular Biology Core), Kira Lathrop (Imaging Core), Katherine Davoli (Histology Core), and the Health Sciences Library System for their advice on RNA sequencing analysis. CLC Genomics Workbench and Ingenuity Pathway Analysis are licensed through the Molecular Biology Information Service of the Health Sciences Library System at the University of Pittsburgh. This study was supported by the National Eye Institute P30-EY008098 (KCC), R21-EY039420 (KCC), P30-EY026877 (JLG), a grant-in-aid from The Glaucoma Foundation (KCC), Gilbert Family Foundation (JLG, YP), Cancer Prevention and Research Institute of Texas (CPRIT; YP), unrestricted grants (JAS, JLG), and a career development award (KCC) from Research to Prevent Blindness, Inc.

These authors contributed equally: Dimitrios Stavropoulos, Chia-Chun Liu. Department of Ophthalmology, UPMC Vision Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Dimitrios Stavropoulos, Chia-Chun Liu, Shining Wang, Chi-Yu Chen, Mishal Rao, Laman Mirzaliyeva, Hui-Chun Cheng, Ya-Ling Lin, Andrew M. Williams, Takaaki Kuwajima, José-Alain Sahel & Kun-Che Chang Department of Translational Neuroscience, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Spencer Center for Vision Research, Byers Eye Institute, Stanford University, Stanford, CA, USA Department of Neurobiology, Center of Neuroscience, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Jeffrey Goldberg and Kun-Che Chang are inventors on a published U.S. patent application related to genetic therapy for glaucoma and optic neuropathies (U.S.