Light-induced retinal damage involves tyrosine 33 phosphorylation, mitochondrial and nuclear translocation of WW domain-containing oxidoreductase in vivo.

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, is a known proapoptotic protein and a candidate tumor suppressor. Stress stimuli activate WOX1 via tyrosine 33 (Tyr33) phosphorylation and translocation to the mitochondria and nuclei in vitro. Here, the potential role of WOX1 in light-induced retinal degeneration in vivo was investigated. WOX1 is expressed primarily in the inner retina at perinatal stages, whereas an enhanced expression of WOX1, along with its Tyr33 phosphorylation (p-WOX1), is shown specifically in the retinal ganglion cells in adults. Prolonged exposure of mature rats to constant, low-intensity light (500 lux) for 1-2 months resulted in substantial death of photoreceptors and the presence of activated microglia, astrocytes and Muller glial in the outer retina. However, the inner retina was not or barely affected. In the damaged inner and outer nuclear layers of rat retina, WOX1 and p-WOX1 were overly expressed. Also, WOX1 colocalized with fragments of opsin-positive cones. In rd mice with an inherited retinal deficiency, upregulation of WOX1 and p-WOX1 in degenerated retina was observed with age. By electron microscopy, a large number of immunogold particles of WOX1 and p-WOX1 were found in the damaged mitochondria and condensed nuclei of degenerating photoreceptors, indicating that WOX1 undergoes activation and translocation to these organelles. In contrast, little or no WOX1-positive particles were found in the Golgi apparatus. In conclusion, activated WOX1 is likely to exert apoptosis of neuronal cells in the outer retina during the light-induced injury and in mice with an inherited retinal defect.

Expression of WW domain-containing oxidoreductase WOX1 in the developing murine nervous system.

WW domain-containing oxidoreductase WOX1, also known as WWOX or FOR, is a proapoptotic protein and a putative tumor suppressor. Hyaluronidases such as PH-20, Hyal-1 and Hyal-2 induce the expression of WOX1, and hyaluronidases and hyaluronan are involved in the embryonic development. In the present study, we document the expression of WOX1 in the developing murine nervous system. Immunohistochemical analysis revealed that WOX1 was differentially expressed in early dividing cells from all three germ layers from embryonic to perinatal stages. In murine fetuses, WOX1 was present prevalently in the brainstem, spinal cord and peripheral nerve bundles, but its expression decreased after birth. In parallel, the expression of WOX1, as determined by Western blotting, was significantly reduced in the brain stem and spinal cord of adult mice. Notably, high levels of WOX1 immunoreactivity was observed in the neural crest-derived structures such as cranial and spinal ganglia and cranial mesenchyme during the late fetal stage. In the adult brain, WOX1 is abundant in the epithelial cells of the choroids plexus and ependymal cells, while a low to moderate level of WOX1 is observed within white matter tracts, such as axonal profiles of the corpus callosum, striatum, optic tract, and cerebral peduncle. WOX1 is shown to mediate apoptosis synergistically with p53 in vitro. Nonetheless, the expression profiles of WOX1 were found to be similar in both p53 wild type and knockout mice, suggesting that WOX1 expression is not controlled by p53-mediated gene transcription. Taken together, in this study we have shown the expression and distribution of WOX1 in developing and adult murine nervous system. The potential role of WOX1 in the neuronal differentiation is discussed.

The retina as a novel in vivo model for studying the role of molecules of the Bcl-2 family in relation to MPTP neurotoxicity.

To determine the roles of different members of the family of B cell lymphoma protooncogene (Bcl-2) in relation to neurotoxin-induced neuronal degeneration, the pattern of the expression of a number of molecules of the Bcl-2 family was studied immunocytochemically in the retinas of C57BL/6J mice after intraperitoneal (IP) injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three days to 12 weeks after MPTP treatment, a detectable reduction of tyrosine hydroxylase immunoreactivity in the amacrine cells was observed, with an increase of Bcl-2 expression in the Müller glial cells, and a de novo expression of Bad and Bax in the retinal ganglion cells, optic nerve fibers and plexiform layers. In contrast, a slight decrease of Bcl-x(L) immunoreactivity in the retinal ganglion cells was observed, whereas Bcl-x(S/L) immunoreactivity was increased slightly in the retinas of MPTP-treated mice compared with that of the controls. In animals that received MPTP injection, an increase in immunostaining of GFAP, glutamine synthetase, and Mac-1 (CD11b) in astrocytes, Müller cells, and microglia was invariably observed, indicating an activation or dysfunction of retinal glial cells. These findings are consistent with the current view that glial dysfunction is important in mediating the cytotoxic effect of a variety of neurotoxic molecules, including MPTP, and that different members of Bcl-2 family may have different roles as far as neuronal degeneration or neuroprotection is concerned.

Alteration of Bcl-2 expression in the nigrostriatal system after kainate injection with or without melatonin co-treatment.

In order to understand further the role of the anti-apoptotic Bcl-2 proto-oncogene protein in excitotoxin-induced brain injury and possible interaction between Bcl-2 and the antioxidant melatonin, the expression of Bcl-2 in various brain parts was studied after intrastriatal injection of kainate (KA, 2.5 nmol) with or without co-treatment of melatonin (10 mg/kg, intraperitoneally (i.p.)). Three days after unilateral injection of KA to the striatum in the rat, a dramatic direct cytotoxic effect was observed, as indicated an expression of Bcl-2 immunoreactivity in TUNEL- and OX-42-positive cells in the KA-injected striatum and traumatized cortical region. A less severe detrimental effect was also observed in the ipsilateral substantia nigra and peritraumatic cortex, as reflected by an upregulation of Bcl-2-immunostained neurons. Surprisingly, a reduction in Bcl-2-immunoreactive neurons that was accompanied by a less severe loss of tyrosine hydroxylase-immunoreactive neurons in the nigrostriatal pathway was observed after co-treatment with melatonin. Western blot analysis confirmed that Bcl-2 expression is elevated in striatum and cortex on the lesioned side, and that its expression was attenuated substantially after systemic administration of melatonin. The results showing an upregulation of Bcl-2 in nigral neurons and reactive microglia after KA lesion are consistent with the view that Bcl-2 is protective in function in the central nervous system.

Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2-virus-infected human liver cells.

The liver has been suspected to be one of the major targets of dengue virus infection. Here, we detected increasing secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), which functions to recruit the immune cells, in dengue-virus-infected liver cells and patients. Three luciferase reporter genes with various deletions at the 5'-end of the RANTES promoter were constructed to explore the RANTES activation mechanism in human liver cells. The reporter gene was optimally activated by dengue-2 virus when the RANTES promoter contains the region from the transcription starting site (+1) to the nucleotide at the -181 position. NF-IL-6 and an undefined factor forming DNA-protein complexes in the RANTES promoter E and A/B regions in the infected cells were demonstrated by electrophoretic mobility shift assay. Further analysis showed that oxidative stress was an upstream inducer of NF-IL-6 and RANTES signaling in dengue-virus-infected liver cells. This finding was demonstrated by three antioxidants (N-acetyl-l-cysteine, nitro-l-arginine methyl ester, and pyrrolidine dithiocarbamate) used to suppress the activation. In contrast, the DNA binding activity of the undefined factor was not affected by the antioxidant treatment, indicating the existence of an oxidant-independent pathway. We hypothesize that dengue virus infection of the liver cells may trigger both an oxidant-dependent and an oxidant-independent pathway to up-regulate RANTES mRNA expression through activating NF-IL-6 and an undefined factor, respectively. In conclusion, the present study suggests a new direction for the study of liver pathogenesis involving RANTES in host immune responses during dengue virus infection.

The antioxidant melatonin reduces cortical neuronal death after intrastriatal injection of kainate in the rat.

The anti-excitotoxic efficacy of the pineal hormone melatonin was investigated in kainate-injured brains of rats. Kainate (a glutamate-receptor agonist, 2.5 nmol in 1 microl) was directly injected to unilateral striatum. Melatonin (10 mg/kg) was administrated intraperitoneally 1 h before and 1, 3, and 5 h after intrastriatal kainate injection in adult Sprague-Dawley rats. Three days after kainate injection, a significant neuronal damage was found, as determined by Nissl staining and the TUNEL method, not only in the injected striatum, but also in the ipsilateral neighboring cortex. The kainate-induced cortical apoptotic neuronal death was significantly attenuated by treatment with melatonin compared with the vehicle control group. However, no detectable changes were observed in the contralateral side of the brain in either vehicle- or melatonin-treated rats. Moreover, the biochemical results indicated that kainate can indeed induce oxidative stress, such as a decrease in the content of total glutathione (GSH), oxidized glutathione (GSSG), and an increase in the ratio of GSSG/GSH in the striatum and cortex compared with the contralateral brain regions. In the kainate-injected striatum, melatonin did not reduce the oxidative stress, but in the neighborhood of injected area-cortex, kainate-induced oxidative stress was significantly reduced by melatonin. Enhancement of glutathione-peroxidase activity was induced by intrastriatal kainate injection, not only in the cortical area of control and melatonin-treated rats, but also in striatum of control rats. However, a large elevation was found in the melatonin-treated cortex. Taking the morphological and biochemical data together, the present results suggest that melatonin functions as an antioxidant by upregulating the glutathione antioxidative defense system, thereby reducing neuronal death caused by excitotoxicity and preventing the kainate-induced damage from spreading to adjacent brain regions.

Rhythms of glutathione peroxidase and glutathione reductase in brain of chick and their inhibition by light.

Melatonin was recently shown to be a component of the antioxidative defense system of organisms due to its free radical scavenging and antioxidant activities. Pharmacologically, melatonin stimulates the activity of the peroxide detoxifying enzyme glutathione peroxidase in rat brain and in several tissues of chicks. In this report, we studied the endogenous rhythm of two antioxidant enzymes, glutathione peroxidase and glutathione reductase, in five regions (hippocampus, hypothalamus, striatum, cortex and cerebellum) of chick brain and correlated them with physiological blood melatonin concentrations. Glutathione peroxidase exhibited a marked 24 h rhythm with peak activity in each brain region which had acrophases about 8 h after lights off and about 4 h after the serum melatonin peak was detected. Glutathione reductase activity exhibited similar robust rhythms with the peaks occurring roughly 2 h after those of glutathione peroxidase. We suggest that neural glutathione peroxidase increases due to the rise of nocturnal melatonin levels while glutathione reductase activity rises slightly later possibly due to an increase of its substrate, oxidized glutathione. The exposure of chicks to constant light for 6 days eliminated the melatonin rhythm as well as the peaks in both glutathione peroxidase and glutathione reductase activities. These findings suggest that the melatonin rhythm may be related to the nighttime increases in the enzyme activities, although other explanations cannot be excluded.

Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen.

Hyperbaric oxygen exposure rapidly induces lipid peroxidation and cellular damage in a variety of organs. In this study, we demonstrate that the exposure of rats to 4 atmospheres of 100% oxygen for 90 min is associated with increased levels of lipid peroxidation products [malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA)] and with changes in the activities of two antioxidative enzymes [glutathione peroxidase (GPX) and glutathione reductase (GR)], as well as in the glutathione status in the lungs and in the brain. Products of lipid peroxidation increased after hyperbaric hyperoxia, both GPX and GR activities were decreased, and levels of total glutathione (reduced+oxidized) and glutathione disulfide (oxidized glutathione) increased in both lung and brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and cerebellum) but not in liver. When animals were injected with melatonin (10 mg/kg) immediately before the 90-min hyperbaric oxygen exposure, all measurements of oxidative damage were prevented and were similar to those in untreated control animals. Melatonin's actions may be related to a variety of mechanisms, some of which remain to be identified, including its ability to directly scavenge free radicals and its induction of antioxidative enzymes via specific melatonin receptors.

Melatonin prevents increases in neural nitric oxide and cyclic GMP production after transient brain ischemia and reperfusion in the Mongolian gerbil (Meriones unguiculatus).

While nitric oxide (NO) has been implicated as a mediator of glutamate excitotoxicity after cerebral ischemia/reperfusion, melatonin has been reported to inhibit brain NO production by suppressing nitric oxide synthase. The purpose of the present studies was to determine the effect of exogenous melatonin administration on NO-induced changes during brain ischemia/reperfusion. Indicators of cerebral cortical and cerebellar NO production [nitrite/nitrate levels and cyclic guanosine monophosphate (cGMP)] were used to estimate neural changes after transient bilateral carotid artery ligation followed by reperfusion in adult Mongolian gerbils (Meriones unguiculatus). Results show for the first time that melatonin prevents the increases in NO and cGMP production after transient ischemia/reperfusion in frontal cerebral cortex and cerebellum of Mongolian gerbils. The inhibitory effect of melatonin on NO production and its ability to scavenge free radicals and the peroxynitrite anion may be responsible for the protective effect of melatonin on neuronal structures during transient ischemia followed by reperfusion.

Effect of melatonin on NF-kappa-B DNA-binding activity in the rat spleen.

It was recently demonstrated that the pineal neurohormone melatonin is a hydroxyl radical scavenger and antioxidant, and that it plays an important role in the immune system. In studies reported herein, we have investigated the relationship of the melatonin level and the NF-kB DNA binding activity in the spleen of Sprague. Dawley rats. These in vivo results indicate that NF-kB DNA binding activity in the spleen is lower at night, when endogenous melatonin levels are elevated, than during the day, when endogenous melatonin levels are lower. Furthermore, exogenously administered melatonin (10 mg/kg) was shown to cause a significant decrease in NF-kB DNA binding activity in the spleen at 60 min after intraperitoneal injection (as compared with vehicle-treated rats). These new findings suggest that the normal night time rise which can be expected for melatonin may be associated with increased NF-kB DNA binding activity in the spleen. The melatonin, therefore, could potentially act to modulate spleen function and/or the immune system by regulating the NF-kB DNA binding activity in the spleen.

Pharmacological effects of melatonin treatment on both locomotor activity and brain serotonin release in rats.

The effects of intraperitoneal administration of pharmacological doses of melatonin (60 mg/kg) on both locomotor activity and brain monoamine release were assessed in rats. The spontaneous levels of either horizontal motion, vertical motion, or total distance traveled were decreased following melatonin injection. On the other hand, the spontaneous levels of postural freezing increased after treatment. External heat exposure (36 degrees C) produced increases in locomotion (including horizontal motion, vertical motion, and total distance traveled) as well as decreases of postural freezing in rats. The heat-induced increases of horizontal motion and total distance traveled as well as decreases of postural freezing were attenuated by melatonin treatment. In addition, cold exposure (4 degrees C) produced increases of vertical motion as well as decreases of postural freezing. Again, the cold-induced behavioral responses were attenuated by melatonin treatment. Biochemical data revealed that the serum levels of melatonin were decreased by both heat and cold exposure in rats. Furthermore, voltammetric data revealed that intraperitoneal administration of melatonin (60 mg/kg) decreased serotonin, but not the dopamine, release in the hypothalamus, the corpus striatum or nucleus accumbens of rat brain. Neither the locomotor activity responses to thermal stress nor brain monoamine release was affected by a smaller dose of melatonin (30 mg/kg, i.p.). The results suggest that systemic administration of melatonin, at pharmacological doses, inhibits brain serotonin release and results in a reduction in both the spontaneous locomotion and the thermal stress-induced locomotor activity responses in rats.

Responses to cold, heat, and pain increase locomotion in rats and are attenuated by pinealectomy.

The effects of pinealectomy on locomotor behavior responses to cold, heat or pain were assessed in freely moving rats. External cold (4 degrees C) or heat (36 degrees C) stress produced increases of locomotion (including horizontal and vertical movement, and total distance traveled), increases of number of turnings (including both clockwise and counterclockwise), and decreases of postural freezing in rats. In addition, pain (produced by intradermal injection of normal saline) was also shown to produce increases of locomotion (including horizontal and vertical movement, and total distance traveled) and decreases of postural freezing in rats. The increases of locomotion (including horizontal and vertical movement, and total distance traveled), as well as the decreases of postural freezing induced by either cold or pain, were attenuated by pretreatment of animals with pinealectomy. The heat-induced increases of vertical movement as well as the decreases of postural freezing were also attenuated by pinealectomy. The results indicate that these nonphotic, stress-provoking stimuli act through the pineal gland to induce escape behaviors to try to get out of the stressed conditions in rats.

Pineal stimulation produces both hypertension and tachycardia in rats.

In anesthetized rats, electrical stimulation of pineal gland elicited proportional hypertension and tachycardia, which could be mimicked by microinjection of an excitatory amino acid, kainic acid (0.3 micrograms), into the pineal gland. The hypertension induced by pineal stimulation was antagonized by either spinal transection or postsynaptic blockade of serotonin receptors, while the tachycardia induced by pineal stimulation was antagonized by either serotonin receptor antagonism, bilateral vagotomy or spinal transection. In addition, postsynaptic blockade of serotonin receptors with cyproheptadine (2-5 mg/Kg, IV) produced both hypotension and bradycardia, while stimulation of 5-HT receptors with DOI (10-250 micrograms/Kg, IV) produced both hypertension and tachycardia in rats. The results indicate that pineal stimulation activates brain 5-HT receptors and results in sympathetic stimulation or parasympathetic inhibition which leads to hypertension and tachycardia in rats.

Melatonin decreases brain serotonin release, arterial pressure and heart rate in rats.

The effects of intravenous administration of melatonin (30-60 mg/kg) or vehicle (10% alcohol) on arterial pressure, heart rate, blood gases or brain serotonin release were assessed in rats under urethane anesthesia. Administration of melatonin, but not the vehicle, produced a dose-related fall in mean arterial pressure, heart rate, or serotonin release in both the corpus striatum and the hypothalamus. Melatonin treatment had an insignificant effect on either PaCO2, PaO2 or pH. In addition, the melatonin-induced depressor responses were abolished by pretreatment with spinal transection, whereas melatonin-induced bradycardia was abolished by pretreatment with bilateral vagotomy. These results suggest that melatonin decreases brain serotonin release and results in sympathetic inhibition or parasympathetic stimulation which leads to hypotension and bradycardia in rats.