Duy-Khanh Dang

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.

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.

Current understanding of methamphetamine-associated dopaminergic neurodegeneration and psychotoxic behaviors

Clinical and preclinical studies have indicated that chronic methamphetamine (MA) use is associated with extensive neurodegeneration, psychosis, and cognitive impairment. Evidence from animal models has suggested a considerable role of excess dopamine or glutamate, oxidative stress, neuroinflammation, and apoptosis in MA-induced neurotoxicity, and that protein kinase Cδ might mediate the interaction among these factors. In addition, the relatively long-lasting and recurrent nature of MA psychosis has been reproduced in animals treated with various dosing regimens of MA, which have shown behavioral sensitization, sociability deficits, and impaired prepulse inhibition. Genetic predisposition as well as dopaminergic and glutamatergic alterations might be important in the development of MA psychosis. Neuroimaging studies have identified functional and morphological changes related to the cognitive dysfunction shown in chronic MA users. Failure in the task-evoked phosphorylation of extracellular signal-related kinase likely underlies MA-induced memory impairment. Recent progress has suggested certain roles of oxidative stress and neuroinflammation in the psychosis and cognitive deficits induced by repeated low doses of MA. This review provides a comprehensive description of pertinent findings from human and animal studies, with an emphasis on the current understanding of the underlying mechanisms of MA neuropsychotoxicity and its relevance to Parkinson’s disease or schizophrenia.

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.

PKCδ-dependent p47 phox activation mediates methamphetamine-induced dopaminergic neurotoxicity

ABSTRACT Protein kinase C (PKC) has been recognized to activate NADPH oxidase (PHOX). However, the interaction between PKC and PHOX in vivo remains elusive. Treatment with methamphetamine (MA) resulted in a selective increase in PKC&dgr; expression out of PKC isoforms. PKC&dgr; co‐immunoprecipitated with p47phox, and facilitated phosphorylation and membrane translocation of p47phox. MA‐induced increases in PHOX activity and reactive oxygen species were attenuated by knockout of p47phox or PKC&dgr;. In addition, MA‐induced impairments in the Nrf‐2‐related glutathione synthetic system were also mitigated by knockout of p47phox or PKC&dgr;. Glutathione‐immunoreactivity was co‐localized in Iba‐1‐labeled microglial cells and in NeuN‐labeled neurons, but not in GFAP‐labeled astrocytes, reflecting the necessity for self‐protection against oxidative stress by mainly microglia. Buthionine‐sulfoximine, an inhibitor of glutathione biosynthesis, potentiated microglial activation and pro‐apoptotic changes, leading to dopaminergic losses. These neurotoxic processes were attenuated by rottlerin, a pharmacological inhibitor of PKC&dgr;, genetic inhibitions of PKC&dgr; [i.e., PKC&dgr; knockout mice (KO) and PKC&dgr; antisense oligonucleotide (ASO)], or genetic inhibition of p47phox (i.e., p47phox KO or p47phox ASO). Rottlerin did not exhibit any additive effects against the protective activity offered by genetic inhibition of p47phox. Therefore, we suggest that PKC&dgr; is a critical regulator for p47phox activation induced by MA, and that Nrf‐2‐dependent GSH induction via inhibition of PKC&dgr; or p47phox, is important for dopaminergic protection against MA insult. Graphical abstract Figure. No caption available. HighlightsInterplay between PKC and PHOX in vivo in the neurotoxic condition remains unclear.PKC&dgr; co‐immunoprecipitated with p47phox and facilitated the potential of p47phox.Methamphetamine‐induced Nrf‐2 system was mitigated by inhibition of p47phox or PKC&dgr;.BSO potentiated microgliosis and apoptotic changes, leading to dopaminergic losses.PKC&dgr; is a critical regulator for p47phox activation induced by methamphetamine.

The role of system Xc− in methamphetamine-induced dopaminergic neurotoxicity in mice

&NA; The cystine/glutamate antiporter (system Xc−, Sxc) transports cystine into cell in exchange for glutamate. Since xCT is a specific subunit of Sxc, we employed xCT knockout mice and investigated whether this antiporter affected methamphetamine (MA)‐induced dopaminergic neurotoxicity. MA treatment significantly increased striatal oxidative burdens in wild type mice. xCT inhibitor [i.e., S‐4‐carboxy‐phenylglycine (CPG), sulfasalazine] or an xCT knockout significantly protected against these oxidative burdens. MA‐induced increases in Iba‐1 expression and Iba‐1‐labeled microglial immunoreactivity (Iba‐1‐IR) were significantly attenuated by CPG or sulfasalazine administration or xCT knockout. CPG or sulfasalazine significantly attenuated MA‐induced TUNEL‐positive cell populations in the striatum of Taconic ICR mice. The decrease in excitatory amino acid transporter‐2 (or glutamate transporter‐1) expression and increase in glutamate release were attenuated by CPG, sulfasalazine or xCT knockout. In addition, CPG, sulfasalazine or xCT knockout significantly protected against dopaminergic loss (i.e., decreases in tyrosine hydroxylase expression and immunoreactivity, and an increase in dopamine turnover rate) induced by MA. However, CPG, sulfasalazine or xCT knockout did not significantly affect the impaired glutathione system [i.e., decrease in reduced glutathione (GSH) and increase in oxidized glutathione (GSSG)] induced by MA. Our results suggest that Sxc mediates MA‐induced neurotoxicity via facilitating oxidative stress, microgliosis, proapoptosis, and glutamate‐related toxicity. HighlightsGenetic or pharmacologic inhibition of xCT exhibits protective potentials against MA.xCT inhibition exerts antioxidant and antiapoptotic effects against MA neurotoxicity.xCT inhibition attenuates microglial activation and glutamate toxicity induced by MA.xCT inhibition does not affect glutathione system impairment induced by MA.

Genetic or pharmacological depletion of cannabinoid CB1 receptor protects against dopaminergic neurotoxicity induced by methamphetamine in mice.

Abstract Accumulating evidence suggests that cannabinoid ligands play delicate roles in cell survival and apoptosis decisions, and that cannabinoid CB1 receptors (CB1R) modulate dopaminergic function. However, the role of CB1R in methamphetamine (MA)‐induced dopaminergic neurotoxicity in vivo remains elusive. Multiple high doses of MA increased phospho‐ERK and CB1R mRNA expressions in the striatum of CB1R (+/+) mice. These increases were attenuated by CB1R antagonists (i.e., AM251 and rimonabant), an ERK inhibitor (U0126), or dopamine D2R antagonist (sulpiride). In addition, treatment with MA resulted in dopaminergic impairments, which were attenuated by CB1R knockout or CB1R antagonists (i.e., AM251 and rimonabant). Consistently, MA‐induced oxidative stresses (i.e., protein oxidation, lipid peroxidation and reactive oxygen species) and pro‐apoptotic changes (i.e., increases in Bax, cleaved PKC&dgr;‐ and cleaved caspase 3‐expression and decrease in Bcl‐2 expression) were observed in the striatum of CB1R (+/+) mice. These toxic effects were attenuated by CB1R knockout or CB1R antagonists. Consistently, treatment with four high doses of CB1R agonists (i.e., WIN 55,212‐2 36 mg/kg and ACEA 16 mg/kg) also resulted in significant oxidative stresses, pro‐apoptotic changes, and dopaminergic impairments. Since CB1R co‐immunoprecipitates PKC&dgr; in the presence of MA or CB1R agonists, we applied PKC&dgr; knockout mice to clarify the role of PKC&dgr; in the neurotoxicity elicited by CB1Rs. CB1R agonist‐induced toxic effects were significantly attenuated by CB1R knockout, CB1R antagonists or PKC&dgr; knockout. Therefore, our results suggest that interaction between D2R, ERK and CB1R is critical for MA‐induced dopaminergic neurotoxicity and that PKC&dgr; mediates dopaminergic damage induced by high‐doses of CB1R agonist. HighlightsMA or CB1R agonists increased CB1R mRNA level in the striatum of wild type mice.Inhibition of CB1R attenuated dopaminergic degeneration induced by MA.CB1R co‐immunoprecipitated PKC&dgr; in the presence of MA or CB1R agonists.Oxidative burdens contributed to PKC&dgr; cleavage and apoptotic activity.CB1R agonists‐induced dopaminergic degeneration was protected by PKC&dgr; inhibition. Graphical abstract Figure. No Caption available.

Growth Hormone-Releaser Diet Attenuates Cognitive Dysfunction in Klotho Mutant Mice via Insulin-Like Growth Factor-1 Receptor Activation in a Genetic Aging Model

Background It has been recognized that a defect in klotho gene expression accelerates the degeneration of multiple age-sensitive traits. Accumulating evidence indicates that aging is associated with declines in cognitive function and the activity of growth hormone (GH)/insulin-like growth factor-1 (IGF-1). Methods In this study, we examined whether a GH-releaser diet could be effective in protecting against cognitive impairment in klotho mutant mice. Results The GH-releaser diet significantly induced the expression of IGF-1 and IGF-1 receptors in the hippocampus of klotho mutant mice. Klotho mutant mice showed significant memory impairments as compared with wild-type mice. In addition, the klotho mutation significantly decreased the expression of cell survival/antiapoptotic factors, including phospho-Akt (p-Akt)/phospho-glycogen synthase kinase3β (p-GSK3β), phospho-extracellular signal-related kinase (p-ERK), and Bcl-2, but significantly increased those of cell death/proapoptotic factors, such as phospho-c-jun N-terminal kinase (p-JNK), Bax, and cleaved caspase-3 in the hippocampus. Treatment with GH-releaser diet significantly attenuated both decreases in the expression of cell survival/antiapoptotic factors and increases in the expression of cell death/proapoptotic factors in the hippocampus of klotho mutant mice. In addition, klotho mutation-induced oxidative stress was significantly attenuated by the GH-releaser diet. Consequently, a GH-releaser diet significantly improved memory function in the klotho mutant mice. GH-releaser diet-mediated actions were significantly reversed by JB-1, an IGF-1 receptor antagonist. Conclusion The results suggest that a GH-releaser diet attenuates oxidative stress, proapoptotic changes and consequent dysfunction in klotho mutant mice by promoting IGF-1 expression and IGF-1 receptor activation.

YY-1224, a terpene trilactone-strengthened Ginkgo biloba, attenuates neurodegenerative changes induced by β-amyloid (1-42) or double transgenic overexpression of APP and PS1 via inhibition of cyclooxygenase-2

BackgroundGinkgo biloba has been reported to possess free radical-scavenging antioxidant activity and anti-inflammatory properties. In our pilot study, YY-1224, a terpene trilactone-strengthened extract of G. biloba, showed anti-inflammatory, neurotrophic, and antioxidant effects.ResultsWe investigated the pharmacological potential of YY-1224 in β-amyloid (Aβ) (1-42)-induced memory impairment using cyclooxygenase-2 (COX-2) knockout (−/−) and APPswe/PS1dE9 transgenic (APP/PS1 Tg) mice. Repeated treatment with YY-1224 significantly attenuated Aβ (1-42)-induced memory impairment in COX-2 (+/+) mice, but not in COX-2 (−/−) mice. YY-1224 significantly attenuated Aβ (1-42)-induced upregulation of platelet-activating factor (PAF) receptor gene expression, reactive oxygen species, and pro-inflammatory factors. In addition, YY-1224 significantly inhibited Aβ (1-42)-induced downregulation of PAF-acetylhydrolase-1 (PAF-AH-1) and peroxisome proliferator-activated receptor γ (PPARγ) gene expression. These changes were more pronounced in COX-2 (+/+) mice than in COX-2 (−/−) mice. YY-1224 significantly attenuated learning impairment, Aβ deposition, and pro-inflammatory microglial activation in APP/PS1 Tg mice, whereas it significantly enhanced PAF-AH and PPARγ expression. A preferential COX-2 inhibitor, meloxicam, did not affect the pharmacological activity by YY-1224, suggesting that the COX-2 gene is a critical mediator of the neuroprotective effects of YY-1224. The protective activity of YY-1224 appeared to be more efficacious than a standard G. biloba extract (Gb) against Aβ insult.ConclusionsOur results suggest that the protective effects of YY-1224 against Aβ toxicity may be associated with its PAF antagonistic- and PPARγ agonistic-potential as well as inhibition of the Aβ-mediated pro-inflammatory switch of microglia phenotypes through suppression of COX-2 expression.