NMNAT2 and NAD⁺ are Downregulated in Glaucomatous RGCs and Overexpression of NMNAT2 Rescues Glaucomatous Neurodegeneration

Abstract

Purpose : Emerging evidence suggests that redox factor NAD⁺ decline is a hallmark of aging and neurodegenerative diseases. This study tested the hypothesis that modulating glaucomatous RGC intrinsic NMNAT2 levels which is the key enzyme in the NAD⁺ biosynthetic process, is a promising gene therapy for glaucomatous neurodegeneration.

Methods : We first profiled the translatomes of RGCs in naïve and silicone oil-induced ocular hypertension (SOHU)/glaucoma mice by RiboTag mRNA sequencing. NMNATs mRNA levels were then validated in SOHU glaucomatous RGCs by in situ hybridization. NAD⁺ levels in retina and optic nerve were acquired by LC/MS based NAD⁺. RGCs soma and axon survival were evaluated by in vivo optical coherence tomography (OCT) imaging and post-mortem retina wholemounts and ON sections histological analysis. Visual function were evaluated by the pattern electroretinogram (PERG) and optokinetic tracking response (OKR) test.

Results : Deep sequencing of translating mRNAs isolated from RGC ribosomes reveals that all three isoforms of NMNATs can be detected in RGCs but NMNAT2 is the most abundant isoform, which is significantly decreased in glaucomatous RGCs (p < 0.0001). In ON crush neuropathy model, AAV2 mediated RGC-specific overexpression of NMNAT2Δex6 promotes RGC soma and axon survival, comparable with cytNMNAT1 in axonal protection while marginally better in soma protection (p < 0.05). In SOHU glaucoma model, RGC-specific overexpression of NMNAT2Δex6 promotes dramatic survival of both RGC somata and axons possibly by significantly restoring NAD⁺ levels in glaucomatous retinas (p < 0.05) and ONs (p < 0.01). In terms of visual function, the peak-to-trough (P1-N2) amplitude ratio of the SOHU eyes to contralateral eyes in PERG increased significantly after NMNAT2 overexpression in RGCs (p < 0.0001) and also significantly preserves visual acuity of the glaucomatous eyes in OKR test (p < 0.0001).

Conclusions : Taking advantage of our recently developed SOHU mouse glaucoma model and RGC-specific AAV promoter, mSncg, we found decreased NMNAT2 expression in glaucomatous RGCs, which may lead to NAD⁺ decline in RGCs and ONs. We further demonstrated that modulating RGC intrinsic levels of NMNAT2 by an AAV2-mSncg vector represents a potent gene therapy strategy for protecting both RGC somata and axons in traumatic ON injury and SOHU glaucoma model.

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