Corneal Denervation Causes Epithelial Apoptosis Through Inhibiting NAD+ Biosynthesis.

PURPOSE: To determine if trigeminal innervations of the corneal epithelium maintains its integrity and homeostasis through controlling the nicotinamide adenine dinucleotide (NAD) content of this tissue.

METHODS: Corneal denervation of C57BL/6 mice was induced by squeezing the nerve bundles that derive from the trigeminal ganglion and was confirmed by whole-mount corneal nerve staining and the sensation test. The apoptosis of the corneal epithelium was examined by TUNEL assay and annexin V/propidium iodide staining. NAD biosynthesis-related enzymes were analyzed by quantitative PCR, immunofluorescence staining, and Western blotting. FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), exogenous nicotinamide mononucleotide (NMN), and NAD+ were used to evaluate the effect of NAD+ on the apoptosis of cultured corneal epithelial cells and epithelial detachment in denervated mice. Protein expression that related to apoptosis and phosphorylation were analyzed by Western blotting.

RESULTS: The denervated mice showed spontaneous corneal epithelial detachment and cell apoptosis accompanied with impaired epithelial NAD+ contents due to low levels of NAMPT. Similarly, inhibition of NAMPT recapitulated epithelial detachment as in denervated mice and induced apoptosis in cultured corneal epithelial cells. The replenishment of NMN or NAD+ partially slowed down corneal nerve fiber degeneration, reduced the epithelial defect in denervated mice, and improved apoptosis induction in FK866-treated cells by restoring the activation levels of SIRT1, AKT, and CREB.

CONCLUSIONS: Corneal denervation lowered epithelial NAD+ contents through reducing the expression of NAMPT and caused cell apoptosis and epithelial defects, suggesting that corneal innervations contribute to epithelial homeostasis by regulating NAD+ biosynthesis.