Retinal ganglion cell specific deletion of arginase-2 preserves visual function after optic nerve crush

Abstract

Purpose : We have shown that global deletion of the mitochondrial arginase isoform arginase 2 (A2) limits retinal ganglion cell (RGC) death in various models of retinal neurodegeneration. Herein, we examined the mechanisms of this neuroprotection and show that neuron-specific deletion of A2 is sufficient to prevent optic nerve crush (ONC)-induced neurodegeneration.

Methods : Wild type (WT), A2^-/- global knockout (A2KO), and Calbindin2-specific (Calb2-cre⁺ A2 ^f/f) KO mice on the C57BL/6J background were subjected to ONC. Stress-activated calcium signaling and mitochondrial dysfunction were assessed by changes in activation status of p38 MAPK, calcium/calmodulin-dependent protein kinase II (CAMKII), and the mitochondrial fission marker dynamin-related protein 1 (Drp1), respectively at 6 and 24 h post ONC. Survival of RGC was quantified with NeuN immunostaining of retina flat-mounts at day 14 and TUNEL labeling of retina sections at day 5 post ONC. Pattern ERGs, spectral domain-optical coherence tomography (SD-OCT), and OptoMotry responses (OMR) were measured at day 21 post ONC. Axonal sprouting was determined by anterograde transport of Cholera Toxin B (CTB).

Results : Early increases in A2 expression, phosphorylation of stress-activated proteins p38 and CAMKII, and the mitochondrial fission protein DRP-1 at ⁶¹⁶Ser, (n=4-7; p<0.05) were detected in WT retinas, and this was abrogated by deletion of A2. IHC showed increased A2 and phospho p38 immunoreactivities within RGC of WT mice. Further, the number of NeuN positive RGC was significantly reduced in A2 ^f/f mice post ONC (39% of sham controls). This RGC loss was reduced in Calb2 A2 KO mice (p=0.07; n=5). Calb2 A2 KO mice also showed fewer TUNEL positive RGC neurons compared to A2 ^f/f (p=0.02; n=4). PERG and visual acuity were decreased in A2 ^f/f mice and improved in Calb2 A2 KO mice (n=4-7; p<0.05). OCT and CTB analysis of retinal thickness and axonal sprouting, respectively, showed improved responses in the Calb2 A2 KO mice after ONC as compared with the A2 ^f/f (n=4-7; p<0.05).

Conclusions : Our data indicate that A2 is neurotoxic in RGCs after injury, and deletion of A2 in Calb2 expressing RGCs limits ONC-induced retinal neurodegeneration and thereby improves visual function. Based on our preliminary findings, stress-activated Calcium signaling via CaMKII/p38 and mitochondrial dysfunction are probable mechanisms involved in A2-mediated RGC cell death.

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