Axonal Protection by Nicotinamide Riboside via SIRT1-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) synthesis pathway has been involved in many biological functions. Nicotinamide riboside (NR) is widely used as an NAD⁺ precursor and known to increase NAD⁺ level in several tissues. The present study aimed to examine the effect of NR on tumor necrosis factor (TNF)-induced optic nerve degeneration and to investigate whether it alters SIRT1 expression and autophagic status in optic nerve. We also examined the localization of nicotinamide riboside kinase 1 (NRK1), which is a downstream enzyme for NR biosynthesis pathway in retina and optic nerve. Intravitreal injection of TNF or TNF plus NR was performed on rats. The p62 and LC3-II protein levels were examined to evaluate autophagic flux in optic nerve. Immunohistochemical analysis was performed to localize NRK1 expression. Morphometric analysis showed substantial axonal protection by NR against TNF-induced axon loss. TNF-induced increment of p62 protein level was significantly inhibited by NR administration. NR administration alone significantly increased the LC3-II levels and reduced p62 levels compared with the basal levels, and upregulated SIRT1 levels in optic nerve. Immunohistochemical analysis showed that NRK1 exists in retinal ganglion cells (RGCs) and nerve fibers in retina and optic nerve. NR administration apparently upregulated NRK1 levels in the TNF-treated eyes as well as the control eyes. Pre-injection of an SIRT1 inhibitor resulted in a significant increase of p62 levels in the NR plus TNF treatment group, implicating that SIRT1 regulates autophagy status. In conclusion, NRK1 exists in RGCs and optic nerve axons. NR exerted protection against axon loss induced by TNF with possible involvement of upregulated NRK1 and SIRT1-autophagy pathway.

Keywords: Autophagy; NRK1; Nicotinamide riboside; SIRT1; p62.

Fig. 1

Paraphenylene-diamine staining of optic nerve axons 2 weeks after injection. a Control group. b TNF-injected group. c 2 pmol NR + TNF-injected group. d 20 pmol NR + TNF-injected group. e 200 pmol NR + TNF-injected group. Scale bar = 10 μm. (f) Morphometric analysis of axon number. (CTL: n = 5, TNF: n = 5, 2 pmol NR + TNF: n = 6, 20 pmol NR + TNF: n = 7, 200 pmol NR + TNF: n = 7) (P^# < 0.0001 vs. CTL, P^* < 0.0005 vs. TNF, P^** < 0.0001 vs. TNF)

Fig. 2

Immunoblotting in samples from optic nerves 1 week after injection. a Effects of TNF or 200 pmol NR + TNF on LC3-II protein level (CTL: n = 3, TNF: n = 3, TNF + NR: n = 3) (P** < 0.005). (b) Effects of 200 pmol NR on LC3-II protein level. (CTL: n = 3, NR: n = 3) (P* < 0.05)

Fig. 3

Immunoblotting in samples from optic nerves 1 week after injection. a Effects of TNF or 200 pmol NR + TNF on p62 protein level. (CTL: n = 4, TNF: n = 4, TNF + NR: n = 4) (P** < 0.005). b Effects of 200 pmol NR on p62 protein level. (CTL: n = 5, NR: n = 5) (P* < 0.05)

Fig. 4

Immunoblotting in samples from optic nerves 1 week after injection. a Effects of TNF or 200 pmol NR + TNF on SIRT1 protein level. (CTL: n = 4, TNF: n = 4, TNF + NR: n = 4) (P** < 0.005). b Effects of 200 pmol NR on SIRT1 protein level. (CTL: n = 3, NR: n = 3) (P* < 0.05)

Fig. 5

Immunohistochemistry in retina and optic nerve. NRK1-positive cells were colocalized with Thy-1-positive cells in normal retina. NRK1 immunoreactivity was colocalized with neurofilament immunoreactivity in normal retina. A few NRK1 immunoreactivities were colocalized with neurofilament immunoreactivity in normal optic nerve, but a lot of NRK immunopositive fibers were colocalized with neurofilament immunoreactivity in the NR-treated optic nerve. Arrows indicate colocalization. Scale bar = 50 μm

Fig. 6

Immunoblotting in samples from optic nerves 1 week after injection. a Effects of TNF or 200 pmol NR + TNF on NRK1 protein level. (CTL: n = 4, TNF: n = 4, TNF + NR: n = 4) (P* < 0.05). b Effects of 200 pmol NR on NRK1 protein level. (CTL: n = 3, NR: n = 3) (P* < 0.05)

Fig. 7

Immunoblotting in samples from optic nerves 1 week after injection. Effects of EX-527 on p62 protein level in the 200 pmol NR plus TNF-treated group. (pre-injection of DMSO and TNF + NR: n = 4, pre-injection of EX-527 and TNF + NR: n = 4) (P* < 0.05)