Purpose: The present study examines the role of Sox11 in the initial response of retinal ganglion cells (RGCs) to axon damage and in optic nerve regeneration in mouse. Methods: Markers of retinal injury were identified using the normal retina database and optic nerve crush (ONC) database on GeneNetwork² (www.genenetwork.org). One gene, Sox11, was highly upregulated following ONC. We examined the role of this transcription factor, Sox11, following ONC and optic nerve regeneration in mice. In situ hybridization was performed using the Affymetrix 2-plex Quantigene View RNA In Situ Hybridization Tissue Assay System. Sox11 was partially knocked out by intravitreal injection of AAV2-CMV-Cre-GFP in Sox11^f/f mice. Optic nerve regeneration model used Pten knockdown. Mice were perfused and the retinas and optic nerves were dissected and examined for RGC survival and axon growth. Results: Sox11 was dramatically upregulated in the retina following ONC injury. The level of Sox11 message increased by approximately eightfold 2 days after ONC. In situ hybridization demonstrated low-level Sox11 message in RGCs and cells in the inner nuclear layer in the normal retina as well as a profound increase in Sox11 message within the ganglion cells following ONC. In Sox11^f/f retinas, partially knocking out Sox11 significantly increased RGC survival after ONC as compared to the AAV2-CMV-GFP control group; however, it had little effect on the ability of axon regeneration. Combinatorial downregulation of both Sox11 and Pten resulted in a significant increase in RGC survival as compared to Pten knockdown only. When Pten was knocked down there was a remarkable increase in the number and the length of regenerating axons. Partially knocking out Sox11 in combination with Pten deletion resulted in a fewer regenerating axons. Conclusion: Taken together, these data demonstrate that Sox11 is involved in the initial response of the retina to injury, playing a role in the early attempts of axon regeneration and neuronal survival. Downregulation of Sox11 aids in RGC survival following injury of optic nerve axons, while a partial knockout of Sox11 negates the axon regeneration stimulated by Pten knockdown.

Glaucoma is a collection of diseases that lead to an irreversible vision loss due to damage of retinal ganglion cells (RGCs). Although the underlying events leading to RGC death are not fully understood, recent research efforts are beginning to define the genetic changes that play a critical role in the initiation and progression of glaucomatous injury and RGC death. Several genetic and experimental animal models have been developed to mimic glaucomatous neurodegeneration. These models differ in many respects but all result in the loss of RGCs. Assessing transcriptional changes across different models could provide a more complete perspective on the molecular drivers of RGC degeneration. For the past several decades, changes in the retinal transcriptome during neurodegeneration process were defined using microarray methods, RNA sequencing and now single cell RNA sequencing. It is understood that these methods have strengths and weaknesses due to technical differences and variations in the analytical tools used. In this review, we focus on the use of transcriptome-wide expression profiling of the changes occurring as RGCs are lost across different glaucoma models. Commonalities of optic nerve crush and glaucoma-induced neurodegeneration are identified and discussed.

Glaucoma etiology often includes retinal ganglion cell (RGC) death associated with elevated intraocular pressure (IOP). However, even when IOP is managed well, disease can progress. It is thus important to develop therapeutic approaches that directly protect RGCs in an IOP-independent manner. Compromised nicotinamide adenine dinucleotide (NAD⁺) metabolism occurs in neurodegenerative diseases, including models of glaucoma. Here we report testing the protective effects of prophylactically systemically administered nicotinamide riboside (NR), a NAD⁺ precursor, in a mouse model of acute RGC damage (optic nerve crush (ONC)), and in a chronic model of RGC degeneration (ocular hypertension induced by intracameral injection of microbeads). For both models, treatment enhanced RGC survival, assessed by counting cells in retinal flatmounts immunostained for Brn3a+. In the ONC model, treatment preserved RGC function, as assessed by pattern electroretinogram, and suppressed retinal inflammation, as assessed by immunofluorescence staining of retinal fixed sections for glial fibrillary acidic protein (GFAP). This is the first study to demonstrate that systemic treatment with NR is protective in acute and chronic models of RGC damage. The protection is significant and, considering that NR is highly bioavailable in and well-tolerated by humans, may support the proposition of prospective human subject studies.

Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG. The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60–100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to QTLs modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human POAG genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10⁻⁶ for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs. Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury. Glaucoma is a complex group of diseases with several known causal mutations and many known risk factors. One well-known risk factor for developing primary open angle glaucoma is the thickness of the central cornea. The present study leverages a unique blend of systems biology methods using BXD recombinant inbred mice and genome-wide association studies from humans to define a putative molecular link between a phenotypic risk factor (central corneal thickness) and glaucoma. We identified a transcription factor, POU6F2, that is found in the developing retinal ganglion cells and cornea. POU6F2 is also present in a subpopulation of retinal ganglion cells and in stem cells of the cornea. Functional studies reveal that POU6F2 is associated with the central corneal thickness and susceptibility of retinal ganglion cells to injury.