Yunsung Nam

Liposomal melatonin rescues methamphetamine‐elicited mitochondrial burdens, pro‐apoptosis, and dopaminergic degeneration through the inhibition PKCδ gene

We have demonstrated that mitochondrial oxidative damage and PKCδ overexpression contribute to methamphetamine‐induced dopaminergic degeneration. Although it is recognized that antioxidant melatonin is effective in preventing neurotoxicity induced by methamphetamine, its precise mechanism remains elusive. C57BL/6J wild‐type mice exhibited a similar degree of dopaminergic deficit when methamphetamine was administered during light and dark phases. Furthermore, dopaminergic neuroprotection by genetic inhibition of PKCδ during the light phase was comparable to that during the dark phase. Thus, we have focused on the light phase to examine whether melatonin modulates PKCδ‐mediated neurotoxic signaling after multiple high doses of methamphetamine. To enhance the bioavailability of melatonin, we applied liposomal melatonin. Treatment with methamphetamine resulted in hyperthermia, mitochondrial translocation of PKCδ, oxidative damage (mitochondria > cytosol), mitochondrial dysfunction, pro‐apoptotic changes, ultrastructural mitochondrial degeneration, dopaminergic degeneration, and behavioral impairment in wild‐type mice. Treatment with liposomal melatonin resulted in a dose‐dependent attenuation against degenerative changes induced by methamphetamine in wild‐type mice. Attenuation by liposomal melatonin might be comparable to that by genetic inhibition (using PKCδ(−/−) mice or PKCδ antisense oligonucleotide). However, liposomal melatonin did not show any additional protective effects on the attenuation by genetic inhibition of PKCδ. Our results suggest that the circadian cycle cannot be a key factor in modulating methamphetamine toxicity under the current experimental condition and that PKCδ is one of the critical target genes for melatonin‐mediated protective effects against mitochondrial burdens (dysfunction), oxidative stress, pro‐apoptosis, and dopaminergic degeneration induced by methamphetamine.

Nobiletin, a citrus flavonoid, improves cognitive impairment and reduces soluble Aβ levels in a triple transgenic mouse model of Alzheimer's disease (3XTg-AD)

Alzheimers disease (AD), the most common form of dementia among the elderly, is characterized by the progressive decline of cognitive function. Increasing evidence indicates that the production and accumulation of amyloid β (Aβ), particularly soluble Aβ oligomers, is central to the pathogenesis of AD. Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, ameliorates learning and memory impairment in olfactory-bulbectomized mice, amyloid precursor protein transgenic mice, NMDA receptor antagonist-treated mice, and senescence-accelerated mouse prone 8. Here, we present evidence that this natural compound improves cognitive impairment and reduces soluble Aβ levels in a triple transgenic mouse model of AD (3XTg-AD) that progressively develops amyloid plaques, neurofibrillary tangles, and cognitive impairments. Treatment with nobiletin (30 mg/kg) for 3 months reversed the impairment of short-term memory and recognition memory in 3XTg-AD mice. Our ELISA analysis also showed that nobiletin reduced the levels of soluble Aβ1-40 in the brain of 3XTg-AD mice. Furthermore, nobiletin reduced ROS levels in the hippocampus of 3XTg-AD as well as wild-type mice. These results suggest that this natural compound has potential to become a novel drug for the treatment and prevention of AD.

Apocynin prevents mitochondrial burdens, microglial activation, and pro-apoptosis induced by a toxic dose of methamphetamine in the striatum of mice via inhibition of p47phox activation by ERK

BackgroundActivation of NADPH oxidase (PHOX) plays a critical role in mediating dopaminergic neuroinflammation. In the present study, we investigated the role of PHOX in methamphetamine (MA)-induced neurotoxic and inflammatory changes in mice.MethodsWe examined changes in mitogen-activated protein kinases (MAPKs), mitochondrial function [i.e., mitochondrial membrane potential, intramitochondrial Ca2+ accumulation, mitochondrial oxidative burdens, mitochondrial superoxide dismutase expression, and mitochondrial translocation of the cleaved form of protein kinase C delta type (cleaved PKCδ)], microglial activity, and pro-apoptotic changes [i.e., cytosolic cytochrome c release, cleaved caspase 3, and terminal deoxynucleotidyl transferase dUDP nick-end labeling (TUNEL) positive populations] after a neurotoxic dose of MA in the striatum of mice to achieve a better understanding of the effects of apocynin, a non-specific PHOX inhibitor, or genetic inhibition of p47phox (by using p47phox knockout mice or p47phox antisense oligonucleotide) against MA-induced dopaminergic neurotoxicity.ResultsPhosphorylation of extracellular signal-regulated kinases (ERK1/2) was most pronounced out of MAPKs after MA. We observed MA-induced phosphorylation and membrane translocation of p47phox in the striatum of mice. The activation of p47phox promoted mitochondrial stresses followed by microglial activation into the M1 phenotype, and pro-apoptotic changes, and led to dopaminergic impairments. ERK activated these signaling pathways. Apocynin or genetic inhibition of p47phox significantly protected these signaling processes induced by MA. ERK inhibitor U0126 did not exhibit any additional positive effects against protective activity mediated by apocynin or p47phox genetic inhibition, suggesting that ERK regulates p47phox activation, and ERK constitutes the crucial target for apocynin-mediated inhibition of PHOX activation.ConclusionsOur results indicate that the neuroprotective mechanism of apocynin against MA insult is via preventing mitochondrial burdens, microglial activation, and pro-apoptotic signaling process by the ERK-dependent activation of p47phox.

Melatonin Attenuates Memory Impairment Induced by Klotho Gene Deficiency Via Interactive Signaling Between MT2 Receptor, ERK, and Nrf2-Related Antioxidant Potential

Background: We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment. Methods: First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin. Results: Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice. Conclusions: Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential.

Ginsenoside Re protects methamphetamine-induced mitochondrial burdens and proapoptosis via genetic inhibition of protein kinase C δ in human neuroblastoma dopaminergic SH-SY5Y cell lines.

Recently, we have demonstrated that ginsenoside Re protects methamphetamine (MA)‐induced dopaminergic toxicity in mice via genetic inhibition of PKCδ and attenuation of mitochondrial stress. In addition, we have reported that induction of mitochondrial glutathione peroxidase (GPx) is also important for neuroprotection mediated by ginsenoside Re. To extend our knowledge, we examined the effects of ginsenoside Re against MA toxicity in vitro condition using SH‐SY5Y neuroblastoma cells. Treatment with ginsenoside Re resulted in significant attenuations against a decrease in the activity of GPx and an increase in the activity of superoxide dismutase (SOD) in the cytosolic and mitochondrial fraction. The changes in glutathione (GSH) paralleled those in GPx in the same experimental condition. Consistently, ginsenoside Re treatment exhibited significant protections against cytosolic and mitochondrial oxidative damage (i.e. lipid peroxidation and protein oxidation), mitochondrial translocation of PKCδ, mitochondrial dysfunction (mitochondrial transmembrane potential and intra‐mitochondrial Ca2+), apoptotic events [i.e., cytochrome c release from mitochondria, cleavage of caspase‐3 and poly(ADP‐ribose)polymerase‐1, nuclear condensation, terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL)‐positive apoptotic cells], and a reduction in the tyrosine hydroxylase (TH) expression and TH activity induced by MA in SH‐SY5Y neuroblastoma cells. These protective effects of ginsenoside Re were comparable to those of PKCδ antisense oligonucleotide (ASO). However, ginsenoside Re did not significantly provide additional protective effects mediated by genetic inhibition of PKCδ. Our results suggest that PKCδ is a specific target for ginsenoside Re‐mediated protective activity against MA toxicity in SH‐SY5Y neuroblastoma cells. Copyright

Repeated exposure to far infrared ray attenuates acute restraint stress in mice via inhibition of JAK2/STAT3 signaling pathway by induction of glutathione peroxidase-1

Exposure to far-infrared ray (FIR) has been shown to exert beneficial effects on cardiovascular and emotional disorders. However, the precise underlying mechanism mediated by FIR remains undetermined. Since restraint stress induces cardiovascular and emotional disorders, the present study investigated whether exposure to FIR affects acute restraint stress (ARS) in mice. c-Fos-immunoreactivity (IR) was significantly increased in the paraventricular hypothalamic nucleus (PVN) and dorsomedial hypothalamic nucleus (DMH) in response to ARS. The increase in c-Fos-IR parallels that in oxidative burdens in the hypothalamus against ARS. Exposure to FIR significantly attenuated increases in the c-Fos-IR, oxidative burdens and corticosterone level. ARS elicited decreases in GSH/GSSG ratio, cytosolic Cu/Zn-superoxide dismutase (SOD-1), glutathione peroxidase (GPx), and glutathione reductase (GR) activities. FIR-mediated attenuation was particularly observed in ARS-induced decrease in GPx, but not in SOD-1 or GR activity. Consistently, ARS-induced decreases in GPx-1-immunoreactivity in PVN and DMH, and decreases in GPx-1 expression in the hypothalamus were significantly attenuated by FIR. ARS-induced significant increases in phosphorylation of JAK2/STAT3, and nuclear translocation and DNA-binding activity of NFκB were observed in the hypothalamus. Exposure to FIR selectively attenuated phosphorylation of JAK2/STAT3, but did not diminish nuclear translocation and DNA-binding activity of NFκB, suggesting that JAK2/STAT3 constitutes a critical target for FIR-mediated pharmacological potential. ARS-induced increase in c-Fos-IR in the PVN and DMH of non-transgenic mice was significantly attenuated by FIR exposure or JAK2/STAT3 inhibitor AG490. GPx-1 overexpressing transgenic mice significantly protected increases in the c-Fos-IR and corticosterone level induced by ARS. However, neither FIR exposure nor AG490 significantly affected attenuations by genetic overexpression of GPx-1. Moreover, AG490 did not exhibit any additional positive effects against the attenuation by genetic overexpression of GPx-1 or FIR exposure. Our results indicate that exposure to FIR significantly protects ARS-induced increases in c-Fos-IR and oxidative burdens via inhibition of JAK2/STAT3 signaling by induction of GPx-1.

Ceruloplasmin is an endogenous protectant against kainate neurotoxicity.

To determine the role of ceruloplasmin (Cp) in epileptic seizures, we used a kainate (KA) seizure animal model and examined hippocampal samples from epileptic patients. Treatment with KA resulted in a time-dependent decrease in Cp protein expression in the hippocampus of rats. Cp-positive cells were colocalized with neurons or reactive astrocytes in KA-treated rats and epileptic patient samples. KA-induced seizures, initial oxidative stress (i.e., hydroxyl radical formation, lipid peroxidation, protein oxidation, and synaptosomal reactive oxygen species), altered iron status (increasing Fe(2+) accumulation and L-ferritin-positive reactive microglial cells and decreasing H-ferritin-positive neurons), and impaired glutathione homeostasis and neurodegeneration (i.e., Fluoro-Nissl and Fluoro-Jade B staining analyses) were more pronounced in Cp antisense oligonucleotide (ASO)- than in Cp sense oligonucleotide-treated rats. Consistently, Cp ASO facilitated KA-induced lactate dehydrogenase (LDH) release, Fe(2+) accumulation, and glutathione loss in neuron-rich and mixed cultures. However, Cp ASO did not alter KA-induced LDH release or Fe(2+) accumulation in the astroglial culture, but did facilitate impairment in glutathione homeostasis in the same culture. Importantly, treatment with human Cp protein resulted in a significant attenuation against these neurotoxicities induced by Cp ASO. Our results suggest that Cp-mediated neuroprotection occurs via the inhibition of seizure-associated oxidative damage (including impairment in glutathione homeostasis), Fe(2+) accumulation, and alterations in ferritin immunoreactivity. Moreover, interactive modulation between neurons and glia was found to be important for Cp upregulation in the attenuation of epileptic damage in both animals and humans.

YY162 prevents ADHD-like behavioral side effects and cytotoxicity induced by Aroclor1254 via interactive signaling between antioxidant potential, BDNF/TrkB, DAT and NET

Methylphenidate (MP) has become the primary drug of choice for treatment of attention-deficit/hyperactivity disorder (ADHD). However, its psychotropic effects severely hamper long-term clinical use. We evaluated the effects of YY162, which consists of terpenoid-strengthened Ginkgo biloba and ginsenoside Rg3, on the ADHD-like condition induced by Aroclor1254, because both components have been suggested to modulate oxidative stress, dopaminergic neurotransmission, and brain-derived neurotrophic factor (BDNF) signaling, which may be critical targets for understanding the pathogenesis of ADHD. YY162 attenuated the increase in reactive oxygen species (ROS) and decrease in BDNF levels induced by Aroclor1254 in SH-SY5Y neuroblastoma cells. YY162 significantly attenuated Aroclor1254-induced ADHD-like behavior and oxidative stress in ICR mice. Furthermore, YY162 attenuated reductions in p-TrkB, BDNF, dopamine transporter (DAT) and norepinephrine transporter (NET) expression. These attenuating effects of YY162 were comparable to those of MP. Importantly, K252a, a TrkB antagonist, counteracted the protective effects of YY162. Our results suggest that YY162 possesses significant protective activities against ADHD-like conditions with negligible behavioral side effects, and that interactive signaling between antioxidant potential and BDNF/TrkB receptor for the positive modulation of the DAT and NET is important for YY162-mediated neuroprotective activity.

Inhibition of protein kinase (PK) Cδ attenuates methamphetamine-induced dopaminergic toxicity via upregulation of phosphorylation of tyrosine hydroxylase at Ser40 by modulation of protein phosphatase 2A and PKA

Recently, we proposed that inhibition of protein kinase (PK) Cδ may be a useful target for protection against methamphetamine (MA)‐induced dopaminergic toxicity. We demonstrated that treatment with MA resulted in a significant decrease in phosphorylation of tyrosine hydroxylase (TH) at Ser40 in the striatum, but not in the phosphorylation of TH at Ser31. In the present study, treatment with rottlerin (1.5 or 3.0 μg, i.c.v, once a day for 5 days), a PKCδ inhibitor, or a PKCδ antisense oligonucleotide (ASO; 2.5 μg/μl, i.c.v., 3 times) significantly attenuated MA‐induced reductions in the phosphorylation of TH at Ser40 and in the expression of PKA in the striatum of mice. This attenuation was significantly counteracted by H89 (10 or 30 ng, i.c.v., 1 h after the last MA administration), a PKA inhibitor. Treatment with rottlerin or ASO significantly attenuated the MA‐induced increase in protein phosphatase (PP) 2A activity. FTY720 (1 or 5 mg/kg, i.p., 1 h after the last MA administration), a PP2A activator, significantly reversed the recovery in TH phosphorylation mediated by inhibition of PKCδ after MA treatment. Both H89 and FTY720 counteracted the recovery of MA‐induced behavioural impairments induced by PKCδ inhibition. The effects, mediated by rottlerin or ASO in MA‐treated wild‐type mice were comparable with those in MA‐treated PKCδ−/− mice. However, neither inhibition of the mitogen‐activated protein kinase subfamily (extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, p38) nor inhibition of calcium calmodulin kinase II significantly altered PKCδ inhibition‐mediated attenuation of MA‐induced impairment of TH phosphorylation. The results suggest that genetic or pharmacological inhibition of PKCδ requires modulation of PKA expression and/or PP2A activity to attenuate the impairment of TH phosphorylation at Ser40 and behavioural activity induced by MA.

Genetic overexpressing of GPx‐1 attenuates cocaine‐induced renal toxicity via induction of anti‐apoptotic factors

The present study investigates the role of the glutathione peroxidase (GPx)‐1 gene in cocaine‐induced renal damage in mice. Multiple doses of cocaine increased lipid peroxidation, protein oxidation, and glutathione oxidation in the kidney of the non‐transgenic mice (non‐TG mice). The enzymatic activities of GPx and glutathione reductase were significantly decreased in non‐TG mice, whereas superoxide dismutase was increased in the early phase of cocaine exposure. Treatment with cocaine resulted in significant decreases in expression of Bcl‐2 and Bcl‐xl in the kidney of non‐TG mice, which resulted in significant increases in Bax and cleaved‐caspase 3. Consistently, cocaine‐induced tubular epithelial vacuolization and focal tubular necrosis were mainly observed in the proximal tubules in the kidneys of non‐TG mice. These renal pathologic changes were much less pronounced in GPx‐1 TG than in non‐TG mice. These results suggest that the GPx‐1 gene is a protective factor against nephrotoxicity induced by cocaine via interactive modulations between antioxidant and cell survival signaling processes.