Axonal Protection by Oral Nicotinamide Riboside Treatment with Upregulated AMPK Phosphorylation in a Rat Glaucomatous Degeneration Model.

Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD+), has been studied to support human health against metabolic stress, cardiovascular disease, and neurodegenerative disease. In the present study, we investigated the effects of oral NR on axonal damage in a rat ocular hypertension model. Intraocular pressure (IOP) elevation was induced by laser irradiation and then the rats received oral NR of 1000 mg/kg/day daily. IOP elevation was seen 7, 14, and 21 days after laser irradiation compared with the controls. We confirmed that oral NR administration significantly increased NAD+ levels in the retina. After 3-week oral administration of NR, morphometric analysis of optic nerve cross-sections showed that the number of axons was protected compared with that in the untreated ocular hypertension group. Oral NR administration significantly prevented retinal ganglion cell (RGC) fiber loss in retinal flat mounts, as shown by neurofilament immunostaining. Immunoblotting samples from the optic nerves showed that oral NR administration augmented the phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) level in rats with and without ocular hypertension induction. Immunohistochemical analysis showed that some p-AMPK-immunopositive fibers were colocalized with neurofilament immunoreactivity in the control group, and oral NR administration enhanced p-AMPK immunopositivity. Our findings suggest that oral NR administration protects against glaucomatous RGC axonal degeneration with the possible upregulation of p-AMPK.

Axonal Protection by Netarsudil, a ROCK Inhibitor, Is Linked to an AMPK-Autophagy Pathway in TNF-Induced Optic Nerve Degeneration.

Purpose: Netarsudil, a Rho kinase inhibitor with norepinephrine transport inhibitory effect, lowers intraocular pressure, however, its effect on axon damage remains to be elucidated. The aim of the current study was to investigate the effect of netarsudil on TNF-induced axon loss and to examine whether it affects phosphorylated-AMP-activated kinase (p-AMPK) and autophagy in the optic nerve. Methods: Intravitreal administration of TNF or TNF with netarsudil was carried out on rats and quantification of axon number was determined. Electron microscopy determined autophagosome numbers. Localization of p-AMPK expression was examined by immunohistochemistry. The changes in p62, LC3-II, and p-AMPK levels were estimated in the optic nerve by immunoblot analysis. The effect of an AMPK activator A769662 or an AMPK inhibitor dorsomorphin on axon number was evaluated. Results: Morphometric analysis revealed apparent protection by netarsudil against TNF-induced axon degeneration. Netarsudil increased autophagosome numbers inside axons. Netarsudil treatment significantly upregulated optic nerve LC3-II levels in both the TNF-treated eyes and the control eyes. Increased p62 protein level induced by TNF was significantly ameliorated by netarsudil. The netarsudil administration alone lessened p62 levels. Netarsudil significantly upregulated the optic nerve p-AMPK levels. A769662 exhibited obvious axonal protection against TNF-induced damage. A769662 treatment upregulated LC3-II levels and the increment of p62 level induced by TNF was significantly ameliorated by A769662. Immunohistochemical analysis revealed that p-AMPK is present in axons. Netarsudil-mediated axonal protection was significantly suppressed by dorsomorphin administration. Conclusions: Netarsudil upregulated p-AMPK and autophagy. Netarsudil-mediated axonal protection may be associated with upregulated p-AMPK.

Akebia Saponin D prevents axonal loss against TNF-induced optic nerve damage with autophagy modulation.

Akebia Saponin D (ASD), a triterpenoid saponin, was shown to have protective effects in certain neuronal cells. The purpose of the present study was to investigate the possibility of ASD to prevent tumor necrosis factor (TNF)-induced axonal loss and the ASD modulation of the biologic process of autophagy in optic nerves. Rats were given intravitreal administration of TNF, simultaneous administration of 2, 20, or 200 pmol ASD and TNF, or ASD alone. LC3-II and p62 expression, which is a marker of autophagic flux, and phosphorylated p38 (p-p38) expression in optic nerves were examined by immunoblot analysis. Morphometric analysis revealed a significant ameliorated effect of ASD against TNF-induced optic nerve damage. p62 was significantly increased in the optic nerve in TNF-treated eyes, but this increase was totally prevented by ASD. The ASD alone injection showed significant reduction of p62 levels compared with the PBS-treated control eyes. LC3-II was significantly increased by ASD treatment in the TNF-injected eyes. p-p38 was significantly increased in the optic nerve in TNF-treated eyes, but this increase was completely prevented by ASD. The protective effects of ASD may be associated with enhanced autophagy activation and inhibition of p-p38.

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

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.

Axonal protection by a small molecule SIRT1 activator, SRT2104, with alteration of autophagy in TNF-induced optic nerve degeneration.

PURPOSE: To examine the effects of SRT2104, an SIRT1 activator, in optic nerve degeneration induced by TNF and to investigate whether it affects the autophagic status after induction of axonal degeneration. STUDY DESIGN: Experimental. METHODS: Adult male Wistar rats received intravitreal injection of TNF alone, concomitant injection of SRT2104 and TNF, or injection of SRT2104 alone. The autophagic status in the optic nerve was evaluated to examine p62 and LC3-II expression by immunoblot analysis. The effect of SRT2104 on TNF-induced axon loss was determined by counting the number of axons. RESULTS: Intravitreal injection of SRT2104 showed a modest protective tendency in the 2-pmol-treated groups against TNF-induced axon loss, although the tendency was not significant on quantitative analysis. However, significant protective effects were found in the 20- or 200-pmol-treated groups. Injection of SRT2104 alone significantly decreased the p62 levels and increased the LC3-II levels as compared with the basal levels. Similarly, concomitant injection of SRT2104 and TNF significantly decreased the p62 levels and increased the LC3-II levels as compared with the TNF-treated group. Upregulation of SIRT1 expression was observed in the optic nerve after SRT2104 treatment. CONCLUSION: The SIRT1 activator SRT2104 exerts axonal protection in TNF-induced optic nerve degeneration. This effect may be associated with upregulated autophagic status in the optic nerve.

Axonal Protection by Tacrolimus with Inhibition of NFATc1 in TNF-Induced Optic Nerve Degeneration.

Tacrolimus, a calcineurin (CaN) inhibitor, has been used for treatment of refractory allergic ocular disease, although its role in optic nerve degeneration remains to be elucidated. In this study, we investigated whether tacrolimus modulates tumor necrosis factor (TNF)-mediated axonal degeneration and whether it alters nuclear factor of activated T cells (NFATc), a downstream effector of CaN signaling. Immunoblot analysis showed no significant difference in CaNAα protein levels in optic nerve on day 3, 7, or 14 after TNF injection compared with PBS injection. However, a significant increase in NFATc1 protein level was observed in optic nerve 7 days after TNF injection. This increase was negated by simultaneous administration of tacrolimus. Administration of tacrolimus alone did not change the NFATc1 protein level in comparison to that observed after PBS injection. A significant increase in TNF protein level was observed in optic nerve 14 days after TNF injection and this increase was prevented by tacrolimus. Immunohistochemical analysis showed the immunoreactivity of NFATc1 to be increased in optic nerve after TNF injection. This increased immunoreactivity was colocalized with glial fibrillary acidic protein and was suppressed by tacrolimus. Treatment of tacrolimus significantly ameliorated the TNF-mediated axonal loss. These results suggest that tacrolimus is neuroprotective against axon loss in TNF-induced optic neuropathy and that the effect arises from suppression of the CaN/NFATc1 pathway.

Involvement of Beclin­1 in axonal protection by short­term hyperglycemia against TNF­induced optic nerve damage.

Beclin­1 serves a pivotal role in autophagosome formation. A previous study demonstrated that streptozotocin­induced hyperglycemia (HG) ameliorates axonal loss induced by tumor necrosis factor (TNF) with upregulation of autophagy in rats. The aim of present study was to examine whether Beclin­1 is involved in this autophagy machinery. Immunoblot analysis of optic nerves demonstrated that HG upregulated Beclin­1 protein expression when compared with normoglycemia (NG). Intravitreal administration of TNF did not alter the optic nerve Beclin­1 expression in NG nor in HG. Beclin­1 immunoreactivity was revealed to be mainly in astrocytes in optic nerves; however, it was also observed in the neurofilaments of the HG group. Morphometric analysis revealed that HG appeared to have substantial ameliorative effects on axon loss and this ameliorative effect was partially prevented by Beclin­1 small interfering RNA. These results indicated that Beclin­1 may exist in neurons and glia in optic nerves and increased Beclin­1 expression may be at least partially associated with axonal protection by HG.

Axonal Protection by Ripasudil, a Rho Kinase Inhibitor, via Modulating Autophagy in TNF-Induced Optic Nerve Degeneration.

Purpose: The Rho kinase inhibitor ripasudil decreases intraocular pressure, although its role in optic nerve axonal damage should be clarified. We therefore investigated whether ripasudil modulates TNF-induced axonal loss and affects autophagy machinery after the induction of optic nerve degeneration. Methods: Rats were given intravitreal injection of TNF, concomitant injection of ripasudil hydrochloride hydrate and TNF, or ripasudil alone. Axon numbers were counted to evaluate the effects of ripasudil against axon loss. Immunoblot analysis was performed to examine p62 as well as LC3-II expression in optic nerves. Electron microscopy was used to determine autophagosome numbers in axons and glia. Immunogold labeling was performed to evaluate autophagosomes in axons. Results: Ripasudil injected intravitreally resulted in significant neuroprotection against TNF-induced axon loss. Intravitreal TNF injection upregulated p62 in the optic nerve, but ripasudil completely inhibited this increment. The ripasudil alone injection diminished p62 and enhanced LC3-II protein levels significantly compared with baseline. Ripasudil-induced upregulation of LC3-II was seen after TNF injection, and immunohistochemical analysis revealed that LC3 colocalized in nerve fibers. Electron microscopic analysis revealed that autophagosomes were present in axons and glia, although autophagosome numbers increased significantly after ripasudil injection only in axons. Conclusions: These results suggest that ripasudil-enhanced intra-axonal autophagy is at least partly involved in axonal protection.