Copper/zinc chelation by clioquinol reduces spinal cord white matter damage and behavioral deficits in a murine MOG-induced multiple sclerosis model.

The present study aimed to evaluate the therapeutic potential of clioquinol (CQ), a metal chelator, on multiple sclerosis pathogenesis. Experimental autoimmune encephalomyelitis was induced by immunization with myelin oligodendrocyte glycoprotein (MOG(35-55)) in female mice. Three weeks after the initial immunization, demyelination and immune cell infiltration in the spinal cord were analyzed. CQ (30mg/kg) was given by gavage once per day for the entire experimental course. CQ profoundly reduced the daily clinical score and incidence rate of EAE mice. The CQ-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination and reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20 and F4/80 positive cells. CQ also remarkably inhibited EAE-associated BBB disruption and MMP-9 activation. Autophagy contributes to clearance of aggregated proteins in astrocytes and neurons. The present study found that EAE increased the induction of autophagy and CQ further increased this expression. Furthermore, the present study found that post-treatment with CQ also reduced the clinical score of EAE and spinal cord demyelination. These results demonstrate that CQ inhibits the clinical features and neuropathological changes associated with EAE. The present study suggests that transition metals may be involved in several steps of multiple sclerosis pathogenesis.

Recurrent/moderate hypoglycemia induces hippocampal dendritic injury, microglial activation, and cognitive impairment in diabetic rats.

BACKGROUND: Recurrent/moderate (R/M) hypoglycemia is common in type 1 diabetes. Although mild or moderate hypoglycemia is not life-threatening, if recurrent, it may cause cognitive impairment. In the present study, we sought to determine whether R/M hypoglycemia leads to neuronal death, dendritic injury, or cognitive impairment. METHODS: The experiments were conducted in normal and in diabetic rats. Rats were subjected to moderate hypoglycemia by insulin without anesthesia. Oxidative stress was evaluated by 4-Hydroxy-2-nonenal immunostaining and neuronal death was determined by Fluoro-Jade B staining 7 days after R/M hypoglycemia. To test whether oxidative injury caused by NADPH oxidase activation, an NADPH oxidase inhibitor, apocynin, was used. Cognitive function was assessed by Barnes maze and open field tests at 6 weeks after R/M hypoglycemia. RESULTS: The present study found that oxidative injury was detected in the dendritic area of the hippocampus after R/M hypoglycemia. Sparse neuronal death was found in the cortex, but no neuronal death was detected in the hippocampus. Significant cognitive impairment and thinning of the CA1 dendritic region was detected 6 weeks after hypoglycemia. Oxidative injury, cognitive impairment, and hippocampal thinning after R/M hypoglycemia were more severe in diabetic rats than in non-diabetic rats. Oxidative damage in the hippocampal CA1 dendritic area and microglial activation were reduced by the NADPH oxidase inhibitor, apocynin. CONCLUSION: The present study suggests that oxidative injury of the hippocampal CA1 dendritic region by R/M hypoglycemia is associated with chronic cognitive impairment in diabetic patients. The present study further suggests that NADPH oxidase inhibition may prevent R/M hypoglycemia-induced hippocampal dendritic injury.

Oncostatin M induces growth arrest of skeletal muscle cells in G1 phase by regulating cyclin D1 protein level.

Oncostatin M (OSM), an IL-6 family cytokine, either inhibits or enhances the growth of cells depending on cell type. Here, we report that OSM inhibits proliferation of skeletal muscle cells by blocking cell cycle progression from G(1) to S phase. OSM treatment significantly reduced levels of cyclin D1 protein and phosphorylation of retinoblastoma protein (Rb) at Ser-795, a CDK4-specific phosphorylation site. The OSM-induced cyclin D1 reduction correlated with decreased amount of the cyclin D1/p27 Kip1 complex and increased amounts of the CDK2/p27 Kip1 complex, resulting in inhibition of CDK2 activity. Results obtained with lactacystin, a proteasome inhibitor, demonstrated that cyclin D1 reduction occurred through ubiquitin/proteasome proteolysis. In addition, activation of STAT3, but not STAT1, is likely to regulate OSM-induced cyclin D1 reduction. Dominant negative (DN)-STAT3 blocked OSM-induced cyclin D1 reduction, and constitutively active-STAT3 also induced cyclin D1 reduction. These results suggest that OSM arrests skeletal muscle cell growth at the G1/S checkpoint and that this response occurs by an ubiquitin/proteasome-dependent cyclin D1 protein reduction which is regulated by STAT3.

The ubiquitin-interacting motif of 26S proteasome subunit S5a induces A549 lung cancer cell death.

The subunit S5a is a key component for the recruitment of ubiquitinated substrates to the 26S proteasome. When the full-length S5a, the N-terminal half of S5a (S5aN) containing the von Willebrand A (vWA) domain, and the C-terminal half of S5a (S5aC) containing two ubiquitin(Ub)-interacting motifs (UIMs) were ectopically expressed in HEK293 cells, Ub-conjugates accumulated most prominently in S5aC-expressing cells. In addition, S5aC induced A549 lung cancer cell death but not non-cancer BEAS-2B cell death. Similar effects were observed using only S5a-UIMs. Our data therefore suggest that S5a-UIMs can be used as upstream inhibitors of the proteasome pathway.

A mushroom extract Piwep from Phellinus igniarius ameliorates experimental autoimmune encephalomyelitis by inhibiting immune cell infiltration in the spinal cord.

The present study aimed to evaluate the therapeutic potential of a mushroom extract from Phellinus igniarius in an animal model of multiple sclerosis. The medicinal mushroom, Phellinus igniarius, contains biologically active compounds that modulate the human immune system. Experimental autoimmune encephalomyelitis (EAE) was induced by immunization with myelin oligodendrocyte glycoprotein (MOG 35-55) in C57BL/6 female mice. A water-ethanol extract of Phellinus igniarius (Piwep) was delivered intraperitoneally every other day for the entire experimental course. Three weeks after the initial immunization, demyelination and immune cell infiltration in the spinal cord were examined. Piwep injection profoundly decreased the daily incidence rate and clinical score of EAE. The Piwep-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination and infiltration of encephalitogenic immune cells including CD4+ T cells, CD8+ T cells, macrophages, and B cells in the spinal cord. Piwep reduced expression of vascular cell adhesion molecule-1 (VCAM-1) in the spinal cord and integrin-α 4 in the lymph node of EAE mice. Piwep also inhibited proliferation of lymphocytes and secretion of interferon-γ in the lymph node of EAE mice. The results suggest that a mushroom extract, Piwep, may have a high therapeutic potential for ameliorating multiple sclerosis progression.

Phlorotannin-rich Ecklonia cava reduces the production of beta-amyloid by modulating alpha- and gamma-secretase expression and activity.

Beta-amyloid (Aß) is a major pathogenic peptide in Alzheimer's disease (AD) and is generated by the processing of amyloid precursor protein (APP). We have previously reported that the brown algae Ecklonia cava, which has anti-oxidant and anti-inflammatory functions, decreased Aß production and further aggregation in HEK293 cells expressing the APP Swedish mutation. Here, we show the reduction mechanism of Aß production using the butanol extract of Ecklonia cava through the examination of expression and activity of alpha-, beta-, and gamma-secretase. Treatment with the extract resulted in the activation of alpha-secretase with a contrasting decrease in its mRNA and protein expression. This activation was consistent with the translocation of the extract into the plasma membrane of the secretase. Gamma-secretase activity was lowered by E. cava, and this effect may be due to the decreased expression of PSEN1 mRNA and protein. In addition, the basal nuclear location of PSEN1, which may affect chromosome missegregation in neurodegenerative disease, was reduced by the extract, despite the significance of this finding remains unclear. Taken together, these results led us to conclude that E. cava regulated the expression and activity of gamma-secretase and alpha-secretase, leading to a reduction in Aß production by the stable cells. Our data indicate that E. cava is a novel natural-product candidate for AD treatment, although further in vivo studies are needed.

Impairment of autophagic flux promotes glucose reperfusion-induced neuro2A cell death after glucose deprivation.

Hypoglycemia-induced brain injury is a common and serious complication of intensive insulin therapy experienced by Type 1 diabetic patients. We previously reported that hypoglycemic neuronal death is triggered by glucose reperfusion after hypoglycemia rather than as a simple result of glucose deprivation. However, the precise mechanism of neuronal death initiated by glucose reperfusion is still unclear. Autophagy is a self-degradation process that acts through a lysosome-mediated trafficking pathway to degrade and recycle intracellular components, thereby regulating metabolism and energy production. Recent studies suggest that autophagic and lysosomal dysfunction leads to abnormal protein degradation and deposition that may contribute to neuronal death. Here, we focused on the relationship between autophagy and lysosomal dysfunction in hypoglycemia-induced neuronal death. In neuronal cells, glucose reperfusion after glucose deprivation resulted in inhibition of autophagy, which may promote cell death. This cell death was accompanied with activation of caspase3 and the lysosomal proteases cathepsin B and D, which indicated impairment of autophagic flux. Taken together, these results suggest that interplay of autophagy, caspase3 activation and lysosomal proteases serve as a basis for neuronal death after hypoglycemia. Thus, we provide the molecular mechanism of neuronal death by glucose reperfusion and suggest some clues for therapeutic strategies to prevent hypoglycemia-induced neuronal death.

Post-treatment of an NADPH oxidase inhibitor prevents seizure-induced neuronal death.

The present study sought to evaluate the neuroprotective effects of apocynin, an NADPH oxidase assembly inhibitor, on seizure-induced neuronal death. Apocynin, also known as acetovanillone, is a natural organic compound isolated from the root of Canadian hemp (Apocynum cannabium). It has been extensively studied to determine its disease-fighting capabilities and application in several brain insults, such as traumatic brain injury and stroke. Here we tested the hypothesis that post-treatment of apocynin may prevent seizure-induced neuronal death by suppression of NADPH oxidase-mediated superoxide production. Temporal lobe epilepsy (TLE) was induced by intraperitoneal injection of pilocarpine (25mg/kg) in male rats. Apocynin (30mg/kg, i.p.) was injected into the intraperitoneal space two hours after seizure onset. A second injection was performed 24h after seizure. To test whether apocynin inhibits NADPH oxidase activation-induced reactive oxygen species (ROS) production, dihydroethidium (dHEt, 5mg/kg, i.p.) was injected before onset of seizure and ROS production was detected five hours after seizure onset. Neuronal oxidative injury (4HNE), neuronal death (Fluoro Jade-B), blood brain barrier (BBB) disruption (IgG leak), neurotrophil infiltration (MPO) and microglia activation (CD11b) in the hippocampus was evaluated at three days after status epilepticus (SE). Pilocarpine-induced seizure increased p47 immunofluorescence in the plasma membrane of hippocampal neurons at 12h post-insult and apocynin treatment prevented this increase. The present study found that apocynin post-treatment decreased ROS production and lipid peroxidation after seizure and decreased the number of degenerating hippocampal neurons. Apocynin also reduced seizure-induced BBB disruption, neurotrophil infiltration and microglial activation. Taken together, the present results suggest that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce seizure-induced neuronal dysfunction.

Zinc chelation reduces hippocampal neurogenesis after pilocarpine-induced seizure.

Several studies have shown that epileptic seizures increase hippocampal neurogenesis in the adult. However, the mechanism underlying increased neurogenesis after seizures remains largely unknown. Neurogenesis occurs in the subgranular zone (SGZ) of the hippocampus in the adult brain, although an understanding of why it actively occurs in this region has remained elusive. A high level of vesicular zinc is localized in the presynaptic terminals of the SGZ. Previously, we demonstrated that a possible correlation may exist between synaptic zinc localization and high rates of neurogenesis in this area after hypoglycemia. Using a lithium-pilocarpine model, we tested our hypothesis that zinc plays a key role in modulating hippocampal neurogenesis after seizure. Then, we injected the zinc chelator, clioquinol (CQ, 30 mg/kg), into the intraperitoneal space to reduce brain zinc availability. Neuronal death was detected with Fluoro Jade-B and NeuN staining to determine whether CQ has neuroprotective effects after seizure. The total number of degenerating and live neurons was similar in vehicle and in CQ treated rats at 1 week after seizure. Neurogenesis was evaluated using BrdU, Ki67 and doublecortin (DCX) immunostaining 1 week after seizure. The number of BrdU, Ki67 and DCX positive cell was increased after seizure. However, the number of BrdU, Ki67 and DCX positive cells was significantly decreased by CQ treatment. Intracellular zinc chelator, N,N,N0,N-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), also reduced seizure-induced neurogenesis in the hippocampus. The present study shows that zinc chelation does not prevent neurodegeneration but does reduce seizure-induced progenitor cell proliferation and neurogenesis. Therefore, this study suggests that zinc has an essential role for modulating hippocampal neurogenesis after seizure.

Prevention of traumatic brain injury-induced neuronal death by inhibition of NADPH oxidase activation.

The present study aimed to evaluate the therapeutic potential of apocynin, an NADPH oxidase assembly inhibitor, on traumatic brain injury. Rat traumatic brain injury (TBI) was performed using a weight drop model. Apocynin (100mg/kg) was injected into the intraperitoneal space 15 min before TBI. Reactive oxygen species (ROS) in the hippocampal CA3 pyramidal neurons were detected by dihydroethidium (dHEt) at 3h after TBI. Oxidative injury was detected by 4-hydroxy-2-nonenal (4HNE) at 6h after TBI. Blood-brain barrier disruption was detected by IgG extravasation and neuronal death was evaluated with Fluoro Jade-B staining 24h after TBI. Microglia activation was detected by CD11b immunohistochemistry in the hippocampus at 1 week after TBI. ROS production was inhibited by apocynin administration in the hippocampal CA3 pyramidal neurons. This pre-treatment with apocynin decreased the blood-brain barrier disruption, the number of degenerating neurons in the hippocampal CA3 region and microglial activation after TBI. The present study indicates that apocynin pre-treatment prevents TBI-induced ROS production, thus decreasing BBB disruption, neuronal death and microglial activation. Therefore, the present study suggests that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce traumatic brain injury-induced neuronal death.

EAAC1 gene deletion alters zinc homeostasis and enhances cortical neuronal injury after transient cerebral ischemia in mice.

The excitatory amino acids glutamate and cysteine are actively transported into neurons from the extracellular space by the high affinity glutamate transporter EAAC1. The astrocyte glutamate transporters, GLT1 and GLAST, are the primary mediators of glutamate clearance. EAAC1 has a limited role in this function. However, uptake of cysteine into neurons via EAAC1 contributes to neuronal antioxidant function by providing cysteine substrate for glutathione synthesis. Mice in which the EAAC1 gene has been deleted were seen to have enhanced susceptibility to neuronal oxidative stress and developed brain atrophy and cognitive function decline with aging. The aim of the current study was to evaluate if EAAC1 confers protection against ischemic events. Young adult CD-1 wild-type or EAAC1(-/-) mice were subjected to 30 min of bilateral common carotid artery occlusion and evaluated for neuronal death and zinc translocation. The intensity of TSQ fluorescence in the cytoplasm of cortical neurons in the EAAC1(-/-) mice was significantly higher than wild-type mice, indicating that the cortical neurons of EAAC1(-/-) mice contain higher cytoplasmic concentrations of labile (or free) zinc. Zinc translocation into cortical neurons was also enhanced in EAAC1(-/-) mice. Three days after ischemia, Fluoro-Jade B staining revealed that EAAC1(-/-) mice had more than twice as many degenerating neurons as wild-type mice. N-acetylcysteine, a membrane-permeant cysteine pro-drug, normalized basal zinc levels, reduced TSQ (+) neurons and reduced ischemic neuronal death in the EAAC1(-/-) mice when delivered in a pre-treatment fashion. Taken together, this study implicates EAAC1-dependent cysteine uptake as an endogenous source of enhancing antioxidant function and zinc homeostasis in neurons in the ischemic brain.

Prevention of acute/severe hypoglycemia-induced neuron death by lactate administration.

Hypoglycemia-induced cerebral neuropathy can occur in patients with diabetes who attempt tight control of blood glucose and may lead to cognitive dysfunction. Accumulating evidence from animal models suggests that hypoglycemia-induced neuronal death is not a simple result of glucose deprivation, but is instead the end result of a multifactorial process. In particular, the excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) consumes cytosolic nicotinamide adenine dinucleotide (NAD(+)), resulting in energy failure. In this study, we investigate whether lactate administration in the absence of cytosolic NAD(+) affords neuroprotection against hypoglycemia-induced neuronal death. Intraperitoneal injection of sodium L-lactate corrected arterial blood pH and blood lactate concentration after hypoglycemia. Lactate administered without glucose was not sufficient to promote electroencephalogram recovery from an isoelectric state during hypoglycemia. However, supplementation of glucose with lactate reduced neuronal death by ∼80% in the hippocampus. Hypoglycemia-induced superoxide production and microglia activation was also substantially reduced by administration of lactate. Taken together, these results suggest an intriguing possibility: that increasing brain lactate following hypoglycemia offsets the decrease in NAD(+) due to overactivation of PARP-1 by acting as an alternative energy substrate that can effectively bypass glycolysis and be fed directly to the citric acid cycle to maintain cellular ATP levels.

Butanol extract of Ecklonia cava prevents production and aggregation of beta-amyloid, and reduces beta-amyloid mediated neuronal death.

Beta-amyloid (Aß) is a major pathogenic peptide for Alzheimer's disease (AD) and is generated by the processing of amyloid precursor protein (APP). The Aß monomers aggregate into oligomeric and fibrillar forms which have been implicated as the toxic species inducing the neuronal dysfunction. Brown algae Ecklonia cava is known for its anti-oxidant and anti-inflammatory functions. Therefore, we tested the effect of E. cava extract on the production and aggregation of Aß peptides. The butanol extract of E. cava reduced Aß secretion from HEK293 cells expressing APP with Swedish mutation and increased soluble APPα and C-terminal fragment-α (CTFα), of which activity was similar to BACE (ß-site of APP cleaving enzyme) inhibitors. Furthermore, the extract inhibited Aß oligomerization, particularly mid-size oligomer formation, confirmed by the ultrastructural morphology. Congo red, thioflavin T assays, and electron microscopy showed that the extract inhibited Aß fibril formation effectively. Finally, the extract protected primary cortical neurons from various Aß-induced cell deaths, especially oligomer-induced death. Although further study is needed to test the effectiveness of the extract in vivo, our results demonstrate, for the first time, that the butanol extract of E. cava could be used as an anti-Aß agent for AD therapeutics.

Plasma carbonic anhydrase II protein is elevated in Alzheimer's disease.

Carbonic anhydrase (CA) plays a critical role in pH regulation, long-term synaptic transformation, and is associated with mental retardation, Alzheimer's disease (AD), and Down syndrome. There is accumulating evidence that CAII is increased in AD brain. The present study focused on the determination of CAII protein level in blood plasma samples using immunoblot and ELISA methods. We compared plasma from 91 AD patients (average age 74.8 y), 83 persons with amnestic mild cognitive impairment (MCI) (average age 73.7 y), and 113 cognitively normal controls (average age 70.8 y). The plasma level of CAII was significantly increased in AD patients, as compared to control groups. CAII levels were higher in males than females. There was an age-dependent increase of CAII. These results provide further evidence that changes in CAII level may play a role in the pathogenesis of AD.

MEK1 plays contrary stage-specific roles in skeletal myogenic differentiation.

The role of extracellular signal-regulated kinase (ERK) signaling in skeletal myogenesis has been reported to be both stimulatory and inhibitory. We propose that this discrepancy may arise from the stage-specific, different roles of mitogen-activated protein kinase kinase 1 (MEK1). We found that the phosphorylated MEK1 level of differentiating C2C12 cells was low on day 1 (early-stage) and reached a maximum on days 2-3 (mid-stage). Cells treated at early stage with the MEK-specific inhibitors, PD184352 and U0126, reduced both the MHC protein level and MCK promoter activity, demonstrating that high MEK1 activity at the mid-stage is required for myogenic differentiation. In contrast, treatment with the ERK-specific inhibitors, FR180204 and ERK inhibitor I, had no effect. However, because the sustained overexpression of constitutively active MEK1 inhibits myogenic differentiation, we further analyzed the stage-specific role of MEK1 using the Tet-Off expression system. The results demonstrated that myogenic differentiation was inhibited if active MEK1 expression was induced earlier than day 1 in differentiation condition, but stimulated if induced after that, demonstrating that activated MEK1 plays differential roles depending on activation time. In addition, the induction of active MEK1 at 12h enhanced the Id2 protein level, while the induction at 36h resulted in reduction. Thus, MEK1 plays stage-specific and contrary roles in myogenesis, and MEK1 activated at the mid-stage promotes muscle differentiation independent of ERK.

Nicotinamide reduces dopamine in postnatal hypothalamus and causes dopamine-deficient phenotype.

Dopamine is an important neurotransmitter in the human central nervous system and also plays a key role in the development of postnatal brains. We previously reported that nicotinamide, a SIRT1 inhibitor, regulates tyrosine hydroxylase (TH) expression in vitro. To investigate the effect of nicotinamide-mediated TH regulation in vivo, nicotinamide was chronically injected into neonatal mice. Interestingly, nicotinamide-treated mice were smaller in size, and their locomotor activity was reduced. L-DOPA treatment caused hypersensitive locomotor activity that indicates a dopamine-depleted state. These changes seemed to be associated with dopamine metabolism in hypothalamus, since dopamine in hypothalamus was reduced but not in striatum. The present study suggests that the regulation of dopamine metabolism during the postnatal development is important and the underlying molecular mechanisms may be associated with SIRT1 signaling.

SIRT1 regulates tyrosine hydroxylase expression and differentiation of neuroblastoma cells via FOXO3a.

To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.