The role of melatonin in amyloid beta-induced inflammation mediated by inflammasome signaling in neuronal cell lines.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. In addition to amyloid beta (Aß) and tau, neuroinflammation is a crucial element in the etiology of this disease. However, the relevance of inflammasome-induced pyroptosis to AD is unknown. We aimed to clarify whether the anti-inflammatory effects of melatonin could prevent Aß-mediated activation of the inflammasome. We demonstrated that Aß upregulated NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD, and cysteinyl aspartate-specific proteinase caspase (caspase 1) expression in SH-SY5Y neuroblastoma cells, resulting in the release of proinflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin-18 (IL-18) and tumor necrosis factor (TNF-α). Melatonin prevented inflammasome signaling and excessive cytokine release caused by Aß. We found that ethyl 2[(2-chlorophenyl)(hydroxy) methyl]acrylate (INF-4E, NLRP3 and caspase 1 inhibitor) significantly abolished Aß-induced proinflammatory cytokine expression. The increase in cleaved-caspase 1, pro-IL18, and cleaved-IL18 caused by Aß suggested the occurrence of pyroptosis, which was further confirmed by the increased expression of N-terminal gasdermin D (N-GSDMD). Melatonin plays a protective role against Aß-induced inflammation via an inflammasome-associated mechanism that is essential in inducing the active forms of cytokines and pyroptosis. The ability of melatonin to inhibit inflammasome may represent a turning point in the treatment of AD progression.

Cognitive impairment and changes of red blood cell components and serum levels of IL-6, IL-18, and L-tryptophan in methamphetamine abusers.

The deficit in cognitive function is more concerning in methamphetamine (MA) users. The cognitive deficit was suspected to be the consequence of neuroinflammation-induced neurological dysregulation. In addition, activating the key enzyme in the tryptophan metabolic pathway by pro-inflammatory cytokines results in metabolite toxicity, further generating cognitive impairments. However, the evidence for the role of neuroinflammation and tryptophan metabolites involved in MA-induced cognitive deficit needs more conclusive study. OBJECTIVES: This retrospective study aimed to determine blood-inflammatory markers, tryptophan metabolite-related molecules, and cognitive function in MA abusers compared to healthy control (HC) participants. METHODS: The cognitive functions were evaluated using Stroop, Go/No-Go, One Back Task (OBT), and Wisconsin Card Sorting Test-64 (WCST-64). Blood samples were analyzed for complete blood count (CBC) analysis, serum inflammatory cytokines interleukin (IL)-6 and IL-18 and tryptophan metabolites. RESULTS: MA group exhibited poor cognitive performance in selective attention, inhibition, working memory, cognitive flexibility, concept formation and processing speed compared to HC. Reduction in red blood cell (RBC) components but induction in white blood cells (WBCs) and IL-6 were observed in MA abusers, which might indicate anemia of (systemic chronic low-grade) inflammation. In addition, the depletion of precursor in the tryptophan metabolic pathway, L-tryptophan was also observed in MA users, which might represent induction in tryptophan metabolites. CONCLUSION: These findings emphasize that blood biomarkers might be a surrogate marker to predict the role of neuroinflammation and abnormal tryptophan metabolite in MA-induced cognitive impairments.

Characterization of endotoxin free protein production of brain-derived neurotrophic factor (BDNF) for the study of Parkinson model in SH-SY5Y differentiated cells.

Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

Psychometric Assessment of the Handwriting Proficiency Screening Questionnaire (HPSQ)-Thai Version for Primary School-Aged Children.

In this study, the original Handwriting Proficiency Screening Questionnaire (HPSQ) was translated into Thai and cross-culturally adapted for use among school-aged children in Thailand. Additionally, the initial psychometric properties of the new Thai version were assessed, including internal consistency, construct validity, and content validity. The original HPSQ was forward-translated by two independent translators from English to Thai and then back-translated. A final consolidation was conducted by an expert committee to develop the Thai HPSQ. In the psychometric evaluation, content validity was quantified using the item-objective congruence (IOC) value for each item. Intra-rater and inter-rater reliabilities were also assessed. Internal consistency was measured using Cronbach's alpha coefficient, and confirmatory factor analysis models were used to examine its construct validity. The Thai version of the HPSQ had excellent internal consistency (α = 0.92), good construct, and content validity (IOC value > 0.6). Intra-rater reliability was good (intraclass correlation coefficient (ICC) = 0.98), and inter-rater reliability ranged from fair to good (ICC = 0.46−0.77). Factor analysis revealed that a three-factor model best fitted the data. Thus, the Thai version of the HPSQ is a reliable and valid instrument for handwriting evaluation among Thai school-aged children. It can be useful for teachers and therapists to identify students with handwriting problems.

Proteomic profiling reveals neuronal ion channel dysregulation and cellular responses to DNA damage-induced cell cycle arrest and senescence in human neuroblastoma SH-SY5Y cells exposed to cypermethrin.

Cypermethrin (CYP), a synthetic pyrethroid of class II, is widely used as a pesticide worldwide. The primary target of cypermethrin is a voltage-gated sodium channel. The neurotoxicity of CYP has been extensively studied in terms of affecting neuronal development, increasing cellular oxidative stress, and apoptosis. However, little is known about how it affects the expression of channel proteins involved in synaptic transmission, as well as the effects of cypermethrin on DNA damage and cell cycle processes. We found that the ligand and voltage-gated calcium channels and proteins involved in synaptic transmission including NMDA 1 receptor subunit, alpha 1A-voltage-dependent calcium channel, synaptotagmin-17, and synaptojanin-2 were downregulated in CYP-treated cells. After 48 h of CYP exposure, cell viability was reduced with flattened and enlarged morphology. The levels of 23 proteins regulating cell cycle processes were altered in CYP-treated cells, according to a proteomic study. The cell cycle analysis showed elevated G0/G1 cell cycle arrest and DNA fragmentation at the sub-G0 stage after CYP exposure. CYP treatment also increased senescence-associated ß-galactosidase positive cells, DNA damage, and apoptotic markers. Taken together, the current study showed that cypermethrin exposure caused DNA damage and hastened cellular senescence and apoptosis via disrupting cell cycle regulation. In addition, despite its primary target sodium channel, CYP might cause synaptic dysfunction via the downregulation of synaptic proteins and dysregulation of synapse-associated ion channels.

Inorganic arsenic contamination and the health of children living near an inactive mining site: northern Thailand.

Arsenic toxicity is a global health problem affecting millions of people. Contamination is caused by arsenic from natural geological sources leaching into aquifers, contaminating drinking water, and may also be caused by mining and other industrial processes. Acute arsenic poisoning is associated with nausea, vomiting, abdominal pain, and severe diarrhea. Chronic arsenic toxicity results in multisystemic diseases leading to central nervous system (CNS) impairments such as cognitive or intellectual deficits in children. Over the past ten years, arsenic contamination has been reported in northern Thailand. The Ministry of Public Health; Thailand, Forensic Science Institute Thammasat University, and the Research Center to Promote Safety and Prevent Injuries in Children at the Ramathibodi Hospital compiled a report on the health impact of the population within a 10 kilometer radius around a mine tailing in the Phichit, Phitsanulok, and Phetchabun Provinces of Thailand. It showed that more than 30 % of children (aged 8-13 years) had higher than normal arsenic contamination levels based on the Agency for Toxic Substances and Disease Registry (ATSDR). After the publication of that report, the mine was temporarily closed in 2016. Based on this data, this research aimed to follow arsenic contamination after the mining operation had stopped operation for three years. The study showed that 4.5 % of school aged children had levels of inorganic arsenic in their urine, higher than the normal range (ATSDR), showing clearly that inorganic arsenic contamination is still above the normal range in children living near an inactive mining site. Therefore, monitoring heavy metal contamination in Thailand and the health effects on vulnerable children who live near mines during regular operation or after being temporarily suspended can prevent and mitigate possible health impacts.

Melatonin Attenuates Methamphetamine-Induced Alteration of Amyloid ß Precursor Protein Cleaving Enzyme Expressions via Melatonin Receptor in Human Neuroblastoma Cells.

Alzheimer's disease (AD) is the most prominent neurodegenerative disease represented by the loss of memory and cognitive impairment symptoms and is one of the major health imperilments among the elderly. Amyloid (Aß) deposit inside the neuron is one of the characteristic pathological hallmarks of this disease, leading to neuronal cell death. In the amyloidogenic processing, the amyloid precursor protein (APP) is cleaved by beta-secretase and γ-secretase to generate Aß. Methamphetamine (METH) is a psychostimulant drug that causes neurodegeneration and detrimental cognitive deficits. The analogy between the neurotoxic and neurodegenerative profile of METH and AD pathology necessitates an exploration of the underlying molecular mechanisms. In the present study, we found that METH ineluctably affects APP processing, which might contribute to the marked production of Aß in human neuroblastoma cells. Melatonin, an indolamine produced and released by the pineal gland as well as other extrapineal, has been protective against METH-induced neurodegenerative processes, thus rescuing neuronal cell death. However, the precise action of melatonin on METH has yet to be determined. We further propose to investigate the protective properties of melatonin on METH-induced APP-cleaving secretases. Pretreatment with melatonin significantly reversed METH-induced APP-cleaving secretases and Aß production. In addition, pretreatment with luzindole, a melatonin receptor antagonist, significantly prevented the protective effect of melatonin, suggesting that the attenuation of the toxic effect on METH-induced APP processing by melatonin was mediated via melatonin receptor. The present results suggested that melatonin has a beneficial role in preventing Aß generation in a cellular model of METH-induced AD.

Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.

Reprogramming cell fates towards mature cell types are a promising cell supply for treating degenerative diseases. Recently, transcription factors and some small molecules have turned into impressive modulating elements for reprogramming cell fates. Melatonin, a pineal hormone, has neuroprotective functions including neural stem cell (NSC) proliferative and differentiative modulation in both embryonic and adult brain. We developed a protocol that could be implemented in the direct reprogramming of human skin fibroblast towards neural cells by using histone deacetylase (HDAC) inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), c-Jun N-terminal kinase (JNK) inhibitor, rho-associated protein kinase inhibitor (Y-27632), cAMP activator, and melatonin treatment. We found that melatonin enhanced neural-transcription factor genes expressions, including brain-specific homeobox/POU domain protein 2 (BRN2), Achaete-Scute Family BHLH transcription Factor 1 (ASCL1), and Myelin Transcription Factor 1 Like (MYT1L). Melatonin also increased the expression of different neural-specific proteins such as doublecortin (DCX), Sex determining region Y-box 2 (Sox2), and neuronal nuclei (NeuN) compared with other five small molecules (valproic acid (VPA), CHIR99021, Forskolin, 1,9 pyrazoloanthrone (SP600125), and Y-27632) combination in the presence and absence of melatonin. A noticeable upregulation of autophagy proteins (microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1) were seen in the melatonin treatment during the induction period while these were reverted in the presence of L-leucine, an autophagy inhibitor. In addition, the expression of NeuN was also significantly reduced by L-leucine. Collectively, our findings revealed an activation of autophagy during neural induction; melatonin enhanced reprogramming efficiency for neuron induction through the modulation of autophagy activation.

Melatonin Attenuates High Glucose-Induced Changes in Beta Amyloid Precursor Protein Processing in Human Neuroblastoma Cells.

Diabetes mellitus (DM), one of metabolic diseases, has been suggested as a risk factor for Alzheimer's disease (AD). However, how the metabolic pathway activates amyloid precursor protein (APP) processing enzymes then contributes to the increase of amyloid-beta (Aß) production, is not clearly understood. In the present study, we aimed to examine the protective effect of melatonin against hyperglycemia-induced alterations in the amyloidogenic pathway. High concentration of glucose was used to induce hyperglycemia in human neuroblastoma SH-SY5Y cells. We found that 30 mM glucose affected the expression of insulin receptors and glucose transporters, which indicated the disruption of glucose sensing. High glucose induced the activation of the phosphorylated protein kinase B (pAkt)/GSK-3ß signaling pathway and a significant increase in the expression of ß-site beta APP cleaving enzyme (BACE1), presenilin1 (PS1) and Aß42. Pretreatment with melatonin significantly reversed these parameters. We also showed that these effects are similar to those effects in the presence of the GSK-3ß blocker, N-(4-methoxybenyl)-N'-(5-nitro-1,3-thiazol-2-yl) urea (ARA) in glucose-treated hyperglycemic cells. These suggested that melatonin exerted an inhibitory effect on the activation of APP-cleaving enzymes via the GSK-3ß signaling pathway. Pretreatment with luzindole, a melatonin receptor MT1 antagonist, significantly prevented the effect of melatonin on the glucose-induced increase level of APP processing enzymes. This suggested that melatonin attenuated the toxic effect on hyperglycemia involving the amyloidogenic pathway partially mediated via melatonin receptor. Taken together the present results suggested that melatonin has a beneficial role in preventing Aß generation in a cellular model of hyperglycemia-induced DM.

Melatonin protects against methamphetamine-induced Alzheimer's disease-like pathological changes in rat hippocampus.

Methamphetamine (METH) is a psychostimulant drug of abuse. METH use is associated with cognitive impairments and neurochemical abnormalities comparable to pathological changes observed in Alzheimer's disease (AD). These observations have stimulated the idea that METH abusers might be prone to develop AD-like signs and symptoms. Melatonin, the pineal hormone, is considered as a potential therapeutic intervention against AD. We thus conducted the present study to explore potential protective roles of melatonin against METH-induced deficits in learning and memory as well as in the appearance of AD-like pathological changes in METH-treated male Wistar rats. We found that melatonin ameliorated METH-induced cognitive impairments in those rats. Melatonin prevented METH-induced decrease in dopamine transporter (DAT) expression in rat hippocampus. Melatonin reversed METH-induced activation of ß-arrestin2, reduction of phosphorylation of protein kinase B (Akt) and METH-induced excessive activity of glycogen synthase kinase-3ß (GSK3ß). Importantly, melatonin inhibited METH-induced changes in the expression of ß-site APP cleaving enzyme (BACE1), disintegrin and metalloproteinase 10 (ADAM10), and presenilin 1 (PS1), as well as the reduction of amyloid beta (Aß)42 production. Immunofluorescence double-labeling demonstrated that melatonin not only prevented the METH-induced loss of DAT but also prevented METH-induced Aß42 overexpression in the dentate gyrus, CA1, and CA3. Furthermore, melatonin also suppressed METH-induced increase in phosphorylated tau. Significantly, melatonin attenuated METH-induced increase in N-methyl-D-aspartate receptor subtype 2 B (NR2B) protein expression and restored METH-induced reduction of Ca2+/calmodulin-dependent protein kinase II (CaMKII). This suggested that melatonin attenuated the toxic effect of METH on the hippocampus involving the amyloidogenic pathway. Taken together, our data suggest that METH abuse may be a predisposing risk factor for AD and that melatonin could serve as a potential therapeutic agent to prevent METH-induced AD like pathology.

Protective Effects of Melatonin on Methamphetamine-Induced Blood-Brain Barrier Dysfunction in Rat Model.

The specialized brain endothelial cells interconnected by unique junctions and adhesion molecules are distinctive features of the blood-brain barrier (BBB), maintaining the homeostasis of the cerebral microenvironment. This study was designed to investigate the protective effects of melatonin on methamphetamine (METH)-induced alterations of BBB integrity. Wistar rats were randomly distributed into groups and underwent melatonin pretreatment and escalating-high doses of METH treatment. Immunohistochemistry was performed to demonstrate the BBB leakage. Protein and RNA samples were isolated from hippocampal and prefrontal cortical tissues and measured expression levels of molecular markers associated with BBB structural components and inflammatory processes. METH provoked the loss of zonula occludens (ZO)-1, occludin, and claudin-5 tight junction proteins. Furthermore, METH caused an excessive increase in matrix metalloproteinase-9 (MMP-9) enzyme, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) and the increase in NAD(P)H oxidase 2 (NOX2). Melatonin exerted the protective effects by recovering tight junction loss; attenuating excessive MMP-9, NOX2, and cell adhesion molecule expression; and reducing serum albumin in the brain. Our results also showed the protective effects of melatonin against METH neurotoxic profiles, characterized by reactive gliosis: microglia (integrin-αM) and astrocyte (GFAP); an excessive upregulation of primary pro-inflammatory cytokines: interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); activation of neuroinflammatory signaling: nuclear factor-kappa B (NF-κB); and suppression of anti-oxidative signaling: nuclear factor erythroid 2-related factor (Nrf2), that may exacerbate BBB structural impairment. Our results provide insights into the beneficial effects of melatonin against METH-induced BBB disruption and mechanisms that play detrimental roles in BBB impairment by in vivo design.

Melatonin attenuates streptozotocin-induced Alzheimer-like features in hyperglycemic rats.

Diabetes mellitus (DM) is increasingly recognized as a risk for developing of Alzheimer's disease (AD). Accordingly, it has been reported that melatonin level is disturbed in both DM and AD which indicates its involvement in the pathophysiology of these diseases. In this study, the neuroprotective activities and relevant mechanisms of melatonin were evaluated in diabetic rat model. Rats were subcutaneously injected with melatonin (10 mg/kg) for 42 consecutive days. Single dose of streptozotocin (60 mg/kg STZ) was intraperitoneally injected. Morris water maze, Western blot and immunohistochemistry analysis of proteins in the hippocampus were measured. We found that melatonin was effective in protecting against memory impairment and decreased formation of Aß42 peptide and phosphorylated tau in the hippocampus of STZ-treated rats. Melatonin significantly restored the reduction in phospho-insulin receptor ß (p-IRß) and ameliorated the increase of inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) in STZ-treated rats. Furthermore, it restored the phosphorylation of glycogen synthase kinase 3ß (GSK3ß), indicating a decreased activity of GSK3ß. Melatonin prevented amyloidogenic processing of ß-amyloid precursor protein (ßAPP) by significantly inhibited ß-site APP cleaving enzyme (BACE1), presenilin 1 (PS1), and ß-cleaved C-terminal fragment (C99). In conclusion, melatonin ameliorates memory deficits in STZ-induced hyperglycemia rats by restoring insulin signaling pathway which is independent of its effects on blood glucose and insulin levels. Thus, melatonin might be a therapeutic option for helping patients suffering from diabetes and contributed to Alzheimer's disease.

Long-term administration of melatonin attenuates neuroinflammation in the aged mouse brain.

Aging is often accompanied by a decline in cognitive function in conjunction with a variety of neurobiological changes, including neuroinflammation. Melatonin is a key endogenous indoleamine secreted by the pineal gland that plays a crucial role in the regulation of circadian rhythms, is a potent free radical scavenger, has anti-inflammatory activity and serves numerous other functions. However, the role of melatonin in sterile inflammation in the brain has not been fully investigated. In the present study, we investigated the neuroinflammation status in aged mouse brains. The results showed that the protein levels of integrin αM (CD11b), glial fibrillary acidic protein (GFAP), the major pro-inflammatory cytokines (interleukin-1 beta [IL-1ß], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) and phosphor-nuclear factor kappa B (pNFκB) were significantly increased, while N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and brain-derived neurotrophic factor (BDNF) were down-regulated in the hippocampus and prefrontal cortex (PFC) of 22-months-old (aged) mice compared with 2-months-old (young adult) mice. Melatonin was administered in the drinking water to a cohort of the aged mice at a dose of 10 mg/kg/day, beginning at an age of 16 months for 6 months. Our results revealed that melatonin significantly attenuated the alterations in these protein levels. The present study suggests an advantageous role for melatonin in anti-inflammation, and this may lead to the prevention of memory impairment in aging.

The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide.

Brain inflammaging is considered as one of the underlying factors of neurodegenerative diseases. The present study aimed to investigate the effects of melatonin, an endogenous indoleamine mainly synthesized by the pineal gland, on hydrogen peroxide (H2O2)-induced inflammaging state in SH-SY5Y cells. Our data showed that p21Cip1 and p16INK4a, cell cycle arrest markers, and the number of senescence-associated ß-galactosidase (SA-ßgal) staining increased significantly in H2O2-treated cells. Melatonin treatment could reverse this effect. Flow cytometry analysis showed a significantly higher percentage in the G0/G1 phase and a lower proportion in the S phase of H2O2 treated cells. Cells pretreated with H2O2 showed a dramatic decrease in the formation of Ki67 immunoactivity while the treatment with melatonin increased Ki67-positive cell. Both mRNA and protein expression levels of the pro-inflammatory cytokines, interleukin-1ß (IL-1ß), IL-6 and, tumor necrosis factor-α (TNF-α) which were increased after induction with H2O2, could be attenuated by melatonin. In addition, melatonin decreased the phospho-nuclear factor kappa B (pNF-κB) expression and prevented its nuclear translocation, as well as abrogated the reduction of nuclear factor erythroid 2-related factor 2 (Nrf2) in SH-SY5Y cells exposed to H2O2. The present data suggested the importance of melatonin on ameliorating inflammation in SH-SY5Y cells.

Melatonin reverses H2 O2 -induced senescence in SH-SY5Y cells by enhancing autophagy via sirtuin 1 deacetylation of the RelA/p65 subunit of NF-κB.

Autophagy, a degradation mechanism that plays a major role in maintaining cellular homeostasis and diminishes in aging, is considered an aging characteristic. Melatonin is an important hormone that plays a wide range of physiological functions, including the anti-aging effect, potentially via the regulation of the Sirtuin1 (SIRT1) pathway. The deacetylation ability of SIRT1 is important for controlling the function of several transcription factors, including nuclear factor kappa B (NF-ĸB). Apart from inflammation, NF-ĸB can regulate autophagy by inhibiting Beclin1, an initiator of autophagy. Although numerous studies have revealed the role of melatonin in regulating autophagy, very limited experiments have shown that melatonin can increase autophagic activity via SIRT1 in a senescent model. This study focuses on the effect of melatonin on autophagy via the deacetylation activity of SIRT1 on RelA/p65, a subunit of NF-ĸB, to determine whether melatonin can attenuate the aging condition. SH-SY5Y cells were treated with H2 O2 to induce the senescent state. These results demonstrated that melatonin reduced a number of beta-galactosidase (SA-ßgal)-positive cells, a senescent marker. In addition, melatonin increased the protein levels of SIRT1, Beclin1, and LC3-II, a hallmark protein of autophagy, and reduced the levels of acetylated-Lys310 in the p65 subunit of NF-ĸB in SH-SY5Y cells treated with H2 O2 . Furthermore, in the presence of SIRT1 inhibitor, melatonin failed to increase autophagic markers. The present data indicate that melatonin enhances autophagic activity via the SIRT1 signaling pathway. Taken together, we propose that in modulating autophagy, melatonin may provide a therapeutically beneficial role in the anti-aging processes.

Melatonin regulates the transcription of ßAPP-cleaving secretases mediated through melatonin receptors in human neuroblastoma SH-SY5Y cells.

Melatonin is involved in the control of various physiological functions, such as sleep, cell growth and free radical scavenging. The ability of melatonin to behave as an antioxidant, together with the fact that the Alzheimer-related amyloid ß-peptide (Aß) triggers oxidative stress through hydroxyl radical-induced cell death, suggests that melatonin could reduce Alzheimer's pathology. Although the exact etiology of Alzheimer's disease (AD) remains to be established, excess Aß is believed to be the primary contributor to the dysfunction and degeneration of neurons that occurs in AD. Aß peptides are produced via the sequential cleavage of ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) and γ-secretase (PS1/PS2), while α-secretase (ADAM10) prevents the production of Aß peptides. We hypothesized that melatonin could inhibit BACE1 and PS1/PS2 and enhance ADAM10 expression. Using the human neuronal SH-SY5Y cell line, we found that melatonin inhibited BACE1 and PS1 and activated ADAM10 mRNA level and protein expression in a concentration-dependent manner and mediated via melatonin G protein-coupled receptors. Melatonin inhibits BACE1 and PS1 protein expressions through the attenuation of nuclear factor-κB phosphorylation (pNF-κB). Moreover, melatonin reduced BACE1 promoter transactivation and consequently downregulated ß-secretase catalytic activity. The present data show that melatonin is not only a potential regulator of ß/γ-secretase but also an activator of α-secretase expression through the activation of protein kinase C, thereby favoring the nonamyloidogenic pathway over the amyloidogenic pathway. Altogether, our findings suggest that melatonin may be a potential therapeutic agent for reducing the risk of AD in humans.

Melatonin regulates aging and neurodegeneration through energy metabolism, epigenetics, autophagy and circadian rhythm pathways.

Brain aging is linked to certain types of neurodegenerative diseases and identifying new therapeutic targets has become critical. Melatonin, a pineal hormone, associates with molecules and signaling pathways that sense and influence energy metabolism, autophagy, and circadian rhythms, including insulin-like growth factor 1 (IGF-1), Forkhead box O (FoxOs), sirtuins and mammalian target of rapamycin (mTOR) signaling pathways. This review summarizes the current understanding of how melatonin, together with molecular, cellular and systemic energy metabolisms, regulates epigenetic processes in the neurons. This information will lead to a greater understanding of molecular epigenetic aging of the brain and anti-aging mechanisms to increase lifespan under healthy conditions.

1-Methyl-4-phenylpyridinium-induced cell death via autophagy through a Bcl-2/Beclin 1 complex-dependent pathway.

Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson's disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity. Therefore, we hypothesized that the MPP(+) toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP(+) increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP(+) can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.

The mechanism for the neuroprotective effect of melatonin against methamphetamine-induced autophagy.

Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson's disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug-induced neurotoxicity. Therefore, we hypothesized that METH-generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell line as our model system, we found that METH-induced autophagy by inhibiting dissociation of Bcl-2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti-apoptotic protein Bcl-2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl-2 was activated by c-Jun N-terminal kinase 1 (JNK 1), which is upstream of Bcl-2 phosphorylation, to induce Bcl-2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl-2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl-2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.

Melatonin attenuates methamphetamine-induced deactivation of the mammalian target of rapamycin signaling to induce autophagy in SK-N-SH cells.

Methamphetamine (METH) is a commonly abused drug that damages nerve terminals by causing reactive oxygen species (ROS) formation, apoptosis, and neuronal damage. Autophagy, a type of programmed cell death independent of apoptosis, is negatively regulated by the mammalian target of the rapamycin (mTOR) signaling pathway. It is not known, however, whether autophagy is involved in METH-induced neurotoxicity. Therefore, we investigated the effect of METH on autophagy and its upstream regulator, the mTOR signaling pathway. Using the SK-N-SH dopaminergic cell line, we found that METH induces the expression of LC3-II, a protein associated with the autophagosome membrane, in a dose-dependent manner. Moreover, METH inhibits the phosphorylation of mTOR and the action of its downstream target, the eukaryotic initiation factor (eIF)4E-binding protein, 4EBP1. Melatonin, a major secretory product of pineal, is a potent naturally produced antioxidant that acts through various mechanisms to ameliorate the toxic effects of ROS. We found that a pretreatment with melatonin enhances mTOR activity and 4EBP1 phosphorylation and protects against the formation of LC3-II in SK-N-SH cells exposed to METH. This work demonstrates a novel role for melatonin as a neuroprotective agent against METH.