Protective effect of thalidomide against N-methyl-D-aspartate-induced retinal neurotoxicity.

Thalidomide, an inhibitor of tumor necrosis factor-α (TNF-α) production, has been indicated to be useful for many inflammatory and oncogenic diseases. In the present study, we examined whether thalidomide (50 mg/kg/day, p.o.) has a protective effect against N-methyl-D-aspartate (NMDA)-induced retinal neurotoxicity in rats. A morphometric analysis showed that systemic administration of thalidomide protects neural cells in the ganglion cell layer (GCL) in a dose-dependent manner and significantly decreases the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in GCL and in the inner nuclear layer (INL). ELISA showed that thalidomide significantly suppressed the elevation of TNF-α 6 and 24 hr after an NMDA injection. Western blot analysis revealed a significant increase in nuclear factor-κB (NF-κB) p65 level in the retinas treated with NMDA at 24 hr after the injection, but not at 6 or 72 hr. Furthermore, an increase in p-JNK and p-p38 levels was also observed in the retina after NMDA injection. Thalidomide suppressed the increased expressions of NF-κB p65, p-JNK, and p-p38 after NMDA injection. Immunohistochemical analysis showed that thalidomide attenuated NF-κB p65 immunoreactivity in the GCL induced by NMDA treatment. In the NMDA-treated group, translocation of NF-κB p65 from the cytoplasm to the nucleus was detected in TUNEL-positive cells exposed to NMDA treatment. These results suggest new indications for thalidomide against neurodegenerative diseases.

Axonal and cell body protection by nicotinamide adenine dinucleotide in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies have demonstrated the crucial role of nicotinamide adenine dinucleotide (NAD) biosynthesis in axonal protection of motor neurons, but the role of nicotinamide mononucleotide adenylyltransferase 1 and NAD in optic nerve degeneration is unclear. Intravitreal injection of tumor necrosis factor (TNF) induces optic nerve degeneration and subsequent loss of retinal ganglion cells. We found that the levels of nicotinamide mononucleotide adenylyltransferase 1 mRNA and protein and of NAD were significantly decreased in the optic nerve after intravitreal injection of TNF in rats. The concomitant disorganization of microtubules with vacuoles and neurofilament accumulations in the axons were blocked by exogenous NAD treatment. Nicotinamide adenine dinucleotide also prevented TNF-induced axonal loss and delayed retinal ganglion cell loss 2 months after TNF injection. Microglia identified by immunohistochemistry were increased in the optic nerves after TNF injection; this increase was inhibited by NAD treatment. These results suggest that axonal nicotinamide mononucleotide adenylyltransferase 1 and NAD declines are associated with TNF-induced optic nerve axonal degeneration and that axonal protection of NAD may be related to its inhibitory effect on microglial activation.

Axonal protection by brain-derived neurotrophic factor associated with CREB phosphorylation in tumor necrosis factor-alpha-induced optic nerve degeneration.

Brain-derived neurotrophic factor (BDNF) is a potent survival and developmental factor that is regulated by cyclic AMP-response element binding protein (CREB) and has a protective effect against retinal ganglion cell (RGC) death. However, the effect of BDNF on the optic nerve axonal degeneration remains to be examined. In this study, we show that intravitreal injection of tumor necrosis factor (TNF)-alpha induces transient increases in phosphorylated-CREB (p-CREB) and BDNF expression in the optic nerve. Administration of exogenous BDNF further increased the p-CREB and endogenous BDNF level and exerted a neuroprotective effect against TNF-alpha-induced axonal loss. The increases in BDNF mRNA and protein induced by TNF-alpha were inhibited significantly by a CRE decoy oligonucleotide. The protective effect of exogenous BDNF on axons was also inhibited by the CRE decoy oligonucleotide. These results suggest that the protective effect of exogenous BDNF may be associated with increases in CREB phosphorylation and endogenous BDNF in the optic nerve.

Effects of unoprostone on phosphorylated extracellular signal-regulated kinase expression in endothelin-1-induced retinal and optic nerve damage.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been implicated in the development of normal- and high-tension glaucoma. We investigated the effects of unoprostone on extracellular signal-regulated kinase (ERK) in ET-1-induced retinal ganglion cell (RGC) death and optic nerve injury. Our morphometric study showed that intravitreal injection of ET-1 led to cell loss in the RGC layer (RGCL) in 28 days. Western blot analysis showed decreased neurofilament (NF) protein in the optic nerve 28 days after ET-1 injection. In this in vivo model, increased phosphorylated ERK (p-ERK) was observed in the retina on 1 day and subsequently in the optic nerve from 7 days after ET-1 injection. Simultaneous injection of M1, as a metabolite of unoprostone, showed further increased p-ERK levels compared with ET-1 injection alone. Our morphometric study of flat-mount preparations stained with cresyl violet or retrograde labeling with a neuro-tracer and Western blot analysis of NF showed that inhibition of ERK phosphorylation led to acceleration of ET-1-induced RGC death and optic nerve damage. In addition, M1 significantly attenuated both RGC loss and the decrease in NF protein induced by ET-1. The protective effects of M1 were significantly inhibited by U0126, an ERK inhibitor. These results suggest that unoprostone has neuroprotective effects against ET-1-induced neuronal injury through ERK phosphorylation.

Calcium/calmodulin-dependent protein kinase II regulates the phosphorylation of CREB in NMDA-induced retinal neurotoxicity.

We examined the role of the phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and cyclic AMP-response element binding protein (CREB) in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed early elevation of phosphorylated CaMKII (p-CaMKII) protein levels and subsequential elevation of phosphorylated CREB (p-CREB) protein after NMDA injection. Immunohistochemistry showed that p-CaMKII was colocalized with Thy-1-positive retinal ganglion cells (RGCs) after NMDA injection. The increase in the p-CaMKII protein level was significantly inhibited by the preinjection of CaMKII small interfering RNA (siRNA), whereas negative control siRNA did not affect. Moreover, the increase in the p-CREB protein level after NMDA injection was also prevented by preinjection of CaMKII siRNA. In addition, our morphometric study of neurotracer retrograde labeling and Thy-1-positive cells showed that CaMKII siRNA significantly accelerated NMDA-induced RGC loss. Furthermore, the prevention of CREB binding by CRE decoy oligonucleotide also exacerbated RGC loss. These results suggest that the activation of CaMKII may regulate CREB phosphorylation and that the transient phosphorylation of CaMKII and CREB may be a neuroprotective response against NMDA-induced neurotoxicity.