Impact of Neurofibromatosis type 1 (NF1) heterozygosity on RGC death after optic nerve injury

Abstract

Purpose : Neurofibromatosis type 1 (NF1) due to loss of heterozygosity at the neurofibromin/NF1 locus is associated with optic glioma, degeneration of retinal ganglion cells (RGCs), and vision loss. Understanding the impact of heterozygosity at the NF1 locus remains a major goal when considering neuroprotective or restorative therapies. Previous studies using optic nerve crush injury revealed increased RGC death in NF1-heterozygote mice, illustrating a neurofibromin-dependent intracellular signaling pathway responsible for neuronal survival. But there are several types of neurons and glial cells in the retina and optic nerve. Here we asked whether RGC-specific Nf1 heterozygosity increases RGC death after optic nerve injury.

Methods : Nf1 flox/wt mice aged 4-6 weeks under isoflurane anesthesia were injected intravitreally with AAV2-cre to carry out RGC-specific Nf1 one allele deletion, or AAV2-GFP control viral vectors. Two weeks later, mice underwent optic nerve crush conducted by cross-action forceps 2mm posterior to the globe under direct visualization. Two weeks after optic nerve crush, eyes were harvested and post-fixed with 4% PFA for 1h at room temperature. Retinas were isolated, permeabilized with 2% Triton-X-100 in PBS and stained with an RGC-specific anti-RBPMS antibody, followed by mounting on slides and imaging by confocal laser scanning microscopy. Eight images of each retina were imaged and quantified to measure RGC density. Results are presented as cells/mm² for each retina. All experiments were masked.

Results : The survival of RGCs in AAV2-cre-injected eyes was significantly less than that in AAV2-GFP-injected eyes after optic nerve crush (Fig 1.).

Conclusions : RGC-specific Nf1 one allele deletion may exacerbate RGC death after optic nerve injury. Our next step is going to see if astrocyte specific Nf1 heterozygosity using AAV5-GFAP-cre will worsen RGC death after optic nerve injury.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Fig 1. Quantification of RBPMS-positive cells showed a significant difference in RGC numbers after optic nerve crush. Each datapoint represents an individual mouse (n=5). Data were analyzed by Student’s t-test. P=0.0023.