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Akira Murakami, Hiroshi Kuribayashi, Yukihiro Baba, Eisuke Arai, Sumiko Watanabe; Roles of NAD for the survival of retinal progenitors through the regulation of pro-apoptotic gene expression via Sirt-mediated histone acetylation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):597.
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© ARVO (1962-2015); The Authors (2016-present)
NMNAT1 is a nuclear enzyme essential for nicotinamide adenine dinucleotide (NAD) biosynthesis pathways.In recent studies, biallelic mutations in NMNAT1 have been found to cause Lebers congenital amaurosis(LCA), however the mechanisms underlying the LCA pathology nor a role in normal retinal development remain unclear. By loss-of-function analysis in mouse retina, we had reported essential roles of Nmnat1 in retinal progenitor survival. We further asked molecular mechanisms of Nmnat1 and NAD during retinal development.
Loss-of-function of Nmnat1 in retinal development was performed using short hairpin (sh)-RNA-mediated downregulation in mouse retinal explant. Phenotype in proliferation/survival/differentiation was examined by immunostaining of frozen sections. Requirement of related genes was also examined by sh-RNA mediated down-regulation. Histone acetylation was examined by ChIP-qPCR.
A sh-Nmnat1 expression plasmid was introduced into isolated mouse retinas at embryonic day 17 and the retinas were cultured as explants. On day 4, there were large numbers of apoptotic retinal progenitor cells in the inner half of the neuroblastic layer, as well as a decrease in the intracellular NAD content of retinas expressing sh-Nmnat1. The addition of NAD to the culture medium attenuated sh-Nmnat1-induced apoptosis. We then examined effects of downregulation of nuclear proteins, which consume NAD to exert their biological activities, on retinal development. Of the Sirtuin (Sirt) family, the expression of sh-Sirt1 and sh-Sirt6, resulted in a phenotype similar to that of sh-Nmnat1. Sirt proteins are the histone deacetylase, and the expression of sh-Nmnat1 increased the levels of acetylated histones H3/H4. Expression of sh-Nmnat1 resulted in significantly increased expression of Noxa and Fas, two pro-apoptotic genes. Acetylation of the genomic 5’ untranslated regions of Noxa and Fas loci was upregulated by sh-Nmnat1 expression and the co-expression of sh-Noxa or sh-Fas with sh-Nmnat1 reduced the number of apoptotic cells induced by sh-Nmnat1 expression alone.
Our findings demonstrate the importance of the NAD biosynthesis pathway in normal development of the retina through regulation of progenitor survival by suppression of pro-apoptotic genes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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