Conditional ablation of NMNAT1 in the murine retina leads to rapid and severe retinal degeneration likely associated with distinct changes in the retinal metabolome

Abstract

Purpose : Mutations in the NAD-synthetic enzyme nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) are linked to a severe form of the blinding disease Leber’s congenital amaurosis (LCA); however, the specific functions of NMNAT1 in the retina are not well understood. We sought to investigate the role of NMNAT1 in retinal development and maintenance through generation and characterization of a retina-specific NMNAT1 knockout mouse model.

Methods : An NMNAT1 conditional knockout model was generated by crossing a “floxed” NMNAT1 transgenic mouse line (generous gift from Dr. Laura Conforti) with a transgenic mouse line expressing Cre recombinase under a retina-specific promoter (Six3-Cre). Retinal function was analyzed using electroretinography (ERG), and retinal morphology was assessed with light microscopy of hematoxylin and eosin-stained retinal sections. Levels of cellular metabolites were quantified using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis on retinal tissue samples from knockout mice and littermate controls, and data was analyzed using MetaboAnalyst software.

Results : NMNAT1 knockout mice experience dramatic loss of retinal outer and inner nuclear layers (ONL/INL) by postnatal day 30 (P30). Additionally, photoreceptor inner and outer segments fail to develop by P10. Consistent with this observation, mice exhibit extremely attenuated ERG responses by P30. Unexpectedly, the peripheral retina appears less sensitive to NMNAT1 loss than the central retina, and exhibits retention of the ONL, INL, and OPL past P75. Knockout retinae show significant changes in levels of many metabolites including thiamine, UDP-glucose, nicotinamide mononucleotide, N1-methylnicotinamide, and glucose-6 phosphate.

Conclusions : Our results support a crucial function for NMNAT1 in retinal development and maintenance—furthermore, it appears as though the extent of this function varies along a spatial gradient from central to peripheral retina. Metabolic analysis reveals NMNAT1 to be a key player in the retinal metabolome, and deregulation of thiamine levels in retina lacking NMNAT1 suggests a hitherto uncharacterized link between these metabolic pathways in mammals. Overall, this study serves as a solid foundation for further inquiry concerning NMNAT1’s role in LCA and the retina.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.