Proteomic Analysis Reveals the Neurotoxic Effects of Chronic Methamphetamine Self-Administration-Induced Cognitive Impairments and the Role of Melatonin-Enhanced Restorative Process during Methamphetamine Withdrawal.

Cognitive flexibility is a crucial ability in humans that can be affected by chronic methamphetamine (METH) addiction. The present study aimed to elucidate the mechanisms underlying cognitive impairment in mice chronically administered METH via an oral self-administration method. Further, the effect of melatonin treatment on recovery of METH-induced cognitive impairment was also investigated. Cognitive performance of the mice was assessed using an attentional set shift task (ASST), and possible underlying neurotoxic mechanisms were investigated by proteomic and western blot analysis of the prefrontal cortex (PFC). The results showed that mice-administered METH for 21 consecutive days exhibited poor cognitive performance compared to controls. Cognitive deficit in mice partly recovered after METH withdrawal. In addition, mice treated with melatonin during METH withdrawal showed a higher cognitive recovery than vehicle-treated METH withdrawal mice. Proteomic and western blot analysis revealed that METH self-administration increased neurotoxic markers, including disruption to the regulation of mitochondrial function, mitophagy, and decreased synaptic plasticity. Treatment with melatonin during withdrawal restored METH-induced mitochondria and synaptic impairments. These findings suggest that METH-induced neurotoxicity partly depends on mitochondrial dysfunction leading to autophagy-dependent cell death and that the recovery of neurological impairments may be enhanced by melatonin treatment during the withdrawal period.

An expression system of channelrhodopsin-2 driven by a minimal Arc/Arg3.1 promoter and Tet system was developed in human neuroblastoma cells.

Advances in neuroscience have relied on the development of techniques that examine neuronal cell activities. One major challenge involves the limitations in labeling and controlling neuronal activities relating to the cell's activation state. In this study, the modified human codon-optimized channelrhodopsin-2 photoreceptor hChR2(C128S) was integrated into function with inducible gene expression methods and materials: the Tet system and the highly efficient minimum promoter of Arc/Arg3.1. The system successfully expressed the target fusion gene exclusively in activated SH-SY5Y human neuroblastoma cells while maintaining the essential characteristics of ChR2. The expression of the channelrhodopsin construct was observed, while the expression duration was refined by treatment with doxycycline. The optogenetic construct here tested the application of the minimum Arc/Arg3.1 promoter, an advanced immediate-early gene promoter, for the expression of the channelrhodopsin gene. Along with its noninvasive nature, this expression system promises to serve dual functions as a cell activity indicator and cell actuator, creating the possibility for researchers to precisely label cells according to their activation state and control the activities of specific neuronal cell populations.

Expression of nano-ferritin in neuronal cells encompassed by minimal Arc promoter system.

Genetic engineering for neuronal cell activity labeling and neuronal cell activity modulation are invaluable for elucidating the underlying characteristics of the brain and neurons. In this study, ferritin fusion protein (FFP) was combined with Tet expression construct under a modified immediate-early gene (IEG) Arc/Arg3.1 promoter so-called SARE-ArcMin. This expression system is a neuronal activity-dependent expression module for nano-ferritin, a radio/magnetic wave-sensitive protein well-accepted as a potential recombinant neuronal actuator. The system was characterized in transcriptional and translational levels in human neuroblastoma SH-SY5Y cells. The mRNA and protein expression levels of nano-ferritin were significant in the activated neurons suggesting that the activity dependent expression patterns of the ferritin also acted as a neuronal cell activation indicator. The system sufficed the need for precise neuronal cell activity specific expression and demonstrated a platform that suggested the use of the nano-ferritin for the study of neuronal cells.

Cathepsin C modulates myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp4e/- mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE.

Mitochondrial Dynamics Impairment in Dexamethasone-Treated Neuronal Cells.

Dexamethasone is an approved steroid for clinical use to activate or suppress cytokines, chemokines, inflammatory enzymes and adhesion molecules. It enters the brain, by-passing the blood brain barrier, and acts through genomic mechanisms. High levels of dexamethasone are able to induce neuronal cell loss, reduce neurogenesis and cause neuronal dysfunction. The exact mechanisms of steroid, especially the dexamethasone contribute to neuronal damage remain unclear. Therefore, the present study explored the mitochondrial dynamics underlying dexamethasone-induced toxicity of human neuroblastoma SH-SY5Y cells. Neuronal cells treatment with the dexamethasone resulted in a marked decrease in cell proliferation. Dexamethasone-induced neurotoxicity also caused upregulation of mitochondrial fusion and cleaved caspase-3 proteins expression. Mitochondria fusion was found in large proportions of dexamethasone-treated cells. These results suggest that dexamethasone-induced hyperfused mitochondrial structures are associated with a caspase-dependent death process in dexamethasone-induced neurotoxicity. These findings point to the high dosage of dexamethasone as being neurotoxic through impairment of mitochondrial dynamics.

The balance between cathepsin C and cystatin F controls remyelination in the brain of Plp1-overexpressing mouse, a chronic demyelinating disease model.

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp4e/- . During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process.

A Complex Interplay Between Melatonin and RORß: RORß is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor ß (RORß) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORß. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORß, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORß as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORß in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORß could not be established, RORα and RORß gene expressions were increased upon 24 h treatment with µM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORß. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

Melatonin attenuates methamphetamine-induced disturbances in mitochondrial dynamics and degeneration in neuroblastoma SH-SY5Y cells.

Methamphetamine (METH) is a psychostimulant drug that can cause toxicity and degeneration in the brain. The toxicity due to METH involves multiple pathways, including the mitochondrial-dependent death pathway. Several pieces of evidence have emphasized that the fragmentation of mitochondria into smaller structures plays some role in the cell-death process. In this study, we investigated the role of mitochondrial dynamics in METH-induced toxicity in human dopaminergic neuroblastoma SH-SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH-induced toxicity was investigated. Our results show that METH significantly decreased cell viability and increased the levels of the mitochondrial fission protein, Fis1 and the Drp1 oligomer. However, the levels of the mitochondrial fusion proteins OPA1 and Mfn1 did not change in METH-treated cells. Melatonin can reverse the toxic effects of the METH-induced reduction in cell viability and the production of the Fis1 protein and the Drp1 oligomer. Moreover, the morphological alteration of mitochondria was investigated in METH-treated cells in the presence of melatonin using transmission electron microscopy (TEM). At 24 hr after METH exposure, typical cell shrinkage was observed in SH-SY5Y cells. Mitochondria were fragmented into small globular structures in a large proportion of METH-treated cells, but tubular networks of mitochondria were present in large proportions of control-untreated cells and METH-treated cells in the presence of melatonin. The results of the present study demonstrate the potential of melatonin to reduce cell death and restore mitochondrial function in neurons affected by METH-induced toxicity.

Effects of melatonin on nervous system aging: neurogenesis and neurodegeneration.

Neural aging as a progressive loss of function involves central and peripheral post-mitotic neurons and neural stem cells (NSCs). It promotes neurodegeneration, impairs neurogenesis, and can be considered a cause of cognitive impairment and sensory and motor deficits in the elderly. Age-related morphological atrophic changes and cellular alterations are addressed by neural aging mechanisms. Neurogenesis declines during aging through several mechanisms such as an increase in quiescence state, changes in lineage fate, telomerase dysfunction, the failure of the DNA repair system, increased apoptosis, and the impairment of self-renewal. The self-renewal transcriptional factor Sox2 has been correlated with retrotransposon L1 and certain cell-cycle- and epigenetic-related factors, which are sometimes considered age-related factors in NSC aging. As neurogenesis decreases, non-mitotic neurons undergo neurodegeneration by oxidative stress, sirtuin, insulin signaling and mTOR alteration, mitochondrial dysfunction, and protein misfolding and aggregation. As neurodegeneration and impaired neurogenesis promote the nervous system aging process, the identification of neuronal anti-aging is required to raise life expectancy. The role of melatonin in increasing neurogenesis and protecting against neurodegeneration has been investigated. Here, we review nervous system aging that is correlated with mechanisms of neurodegeneration and the impairment of neurogenesis and evaluate the effects of melatonin on these processes.

Melatonin enhances the restoration of neurological impairments and cognitive deficits during drug withdrawal in methamphetamine-induced toxicity and endoplasmic reticulum stress in rats.

Methamphetamine (METH) is a psychostimulant with a very high addiction rate. Prolonged use of METH has been observed as one of the root causes of neurotoxicity. Melatonin (Mel) has been found to have a significant role in METH-induced neurotoxicity. This study aimed to investigate the restorative effect of Mel on behavioral flexibility in METH-induced cognitive deficits. Male Sprague-Dawley rats were randomly assigned to be intraperitoneally injected with saline (control) or Meth at 5 mg/kg for 7 consecutive days. Then, METH injection was withdrawn and rats in each group were subcutaneously injected with saline or Mel at 10 mg/kg for 14 consecutive days. The stereotypic behavioral test and attentional set-shifting task (ASST) were used to evaluate neurological functions and cognitive flexibility, respectively. Rats developed abnormal features of stereotyped behaviors and deficits in cognitive flexibility after 7 days of METH administration. However, post-treatment with Mel for 14 days after METH withdrawal dramatically ameliorated the neurological and cognitive deficits in METH-treated rats. Blood biomarkers indicated METH-induced systemic low-grade inflammation. Moreover, METH-induced endoplasmic reticulum (ER) stress in the prefrontal cortex was diminished by melatonin supplementation. These findings might reveal the therapeutic potential of Mel in METH toxicity-induced neurological and cognitive deficits.

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.

The quantitative analysis of EEG during resting and cognitive states related to neurological dysfunctions and cognitive impairments in methamphetamine abusers.

Several lines of evidence demonstrated the deleterious effect of methamphetamine (MA) on neurological and psychological functions. However, recent evidence on the neurological dysfunctions related to cognitive performance and psychosis in MA abusers needs to be elucidated. Therefore, the present study aimed to investigate the neurological functions using EEG measurement during cognitive tests in MA abusers with (MWP) or without (MWOP) psychosis compared to age-matched normal participants. The quantitative EEG (qEEG) was used to reveal the absolute power in 4 brain-wave frequencies including delta, theta, alpha, and beta waves. The results demonstrated poor attention in both groups of MA abusers. The deficit in mental flexibility was observed in MWP. The deficit in inhibition control and working memory were observed in MWOP. The greater delta, alpha and beta brain waves in multiple brain areas were observed in MWP during the resting (eyes-open) state. The greater alpha wave in multiple brain areas of MWP correlated with poor attention. The greater delta wave and lesser beta wave in the frontal brain correlated with poor inhibition and working memory in MWOP respectively. These findings demonstrated the applicability of EEG to determine neurological dysfunction related to cognitive impairments in MA abusers.

The neuroprotective role of melatonin against methamphetamine toxicity-induced neurotransmission dysregulation and cognitive deficits in rats.

Methamphetamine (MA) is a psychostimulant and addictive substance. Long-term uses and toxic high doses of MA can induce neurotoxicity. The present study aimed to investigate the protective role of melatonin against MA toxicity-induced dysregulation of the neurotransmission related to cognitive function in rats. The adult male Sprague Dawley rats were intraperitoneally injected with 5 mg/kg MA for 7 consecutive days with or without subcutaneously injected with 10 mg/kg melatonin before MA injection. Some rats were injected with saline solution (control) or 10 mg/kg melatonin. MA administration induced reduction in total weight gain, neurotoxic features of stereotyped behaviors, deficits in cognitive flexibility, and significantly increased lipid peroxidation in the brain which diminished in melatonin pretreatment. The neurotoxic effect of MA on glutamate, dopamine and GABA transmitters was represented by the alteration of the GluR1, DARPP-32 and parvalbumin (PV) levels, respectively. A significant decrease in the GluR1 was observed in the prefrontal cortex of MA administration in rats. MA administration significantly increased the DARPP-32 but decreased PV in the striatum. Pretreatment of melatonin can abolish the neurotoxic effect of MA on neurotransmission dysregulation. These findings might reveal the antioxidative role of melatonin to restore neurotransmission dysregulation related to cognitive deficits in MA-induced neurotoxicity.

Spilanthes acmella Murr. ameliorates chronic stress through improving mitochondrial function in chronic restraint stress rats.

Chronic stress is a risk factor for the development of psychiatric illnesses through impairment of the ability to appropriately regulate physiological and behavioral responses, but the molecular events that lead to damage of hippocampal neurons remain unclear. The medicinal herb Spilanthes acmella Murr. has been used as a traditional medicine for various diseases and its extracts exhibit antioxidant activity. The present study explored the molecular signals of mitochondrial dynamics and investigated the beneficial effects of S. acmella Murr. An ethyl acetate extract of this plant was used to assess mitochondrial dynamics in response to chronic restraint stress (CRS) in male Sprague-Dawley rats. The results demonstrated that the S. acmella Murr. extract reduced the expression of mitochondrial fission protein but induced HSP60, MnSOD and ATPsynthase in the hippocampus of the CRS rats. In addition, S. acmella Murr. extract reversed depressive symptoms in the forced swim test. Our findings suggested that S. acmella Murr. extract provides a potential treatment of chronic stress, and that the mechanism is associated with the alleviation of neuronal injury and maintenance of mitochondrial function.

Hydrogen peroxide toxicity induces Ras signaling in human neuroblastoma SH-SY5Y cultured cells.

It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

The protective effect of melatonin on methamphetamine-induced calpain-dependent death pathway in human neuroblastoma SH-SY5Y cultured cells.

Methamphetamine (METH) is a potent psychostimulant drug that may cause neuronal cell degeneration. The underlying mechanisms of METH-induced neuronal toxicity remains poorly understood. In this study, we investigated an important role of calpain-dependent cascades in methamphetamine-induced toxicity in human dopaminergic neuroblastoma SH-SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH-induced calpain-dependent death pathway was also investigated. The results of this study show that METH significantly decreased cell viability and tyrosine hydroxylase phosphorylation in SH-SY5Y cultured cells. Melatonin reversed the toxic effect of METH by inducing cell viability. In addition, melatonin was able to restore the reduction in mitochondrial function and phosphorylation of tyrosine hydroxylase in SH-SY5Y treated cells. An induction of calpain expression and activity but a reduction of calpain inhibitor (calpastatin) protein levels were observed in SH-SY5Y cells treated with METH but these effects were diminished by melatonin. These results implicated calpain-dependent death pathways in the processes of METH-induced toxicity and also indicated that melatonin has the capacity to reverse this toxic effect in SH-SY5Y cultured cells.

Aluminum chloride-induced amyloid ß accumulation and endoplasmic reticulum stress in rat brain are averted by melatonin.

Accumulation of aluminium (Al) in the brain is known to be a toxic insult that result in neurodegenerative diseases and melatonin is known to have neuroprotective role. The present study was designed to investigate the neuroprotective effects of melatonin for aluminium chloride (AlCl3)-induced neurotoxicity in rats. Twelve-week old male Wistar rats were orally received 175 mg/kg AlCl3 with or without 5 mg/kg melatonin intraperitoneal pretreatment. Group 3 intraperitoneally recieved 5 mg/kg melatonin and group 4 rats were orally treated with saline solution for 8 weeks. A series of behavioral tests, biochemical analysis and expression of AD-associated proteins in the brain were determined after 7 weeks of all treatments. Our results indicated that AlCl3 treatment tends to induce memory and cognitive impairment. However, melatonin treatment attenuated amyloid beta (Aß) (1-42) level by decreasing ß-secretase, augmented low-density lipoprotein receptor-related protein 1, and neprilysin protein expression. Moreover, AlCl3 -induced endoplasmic reticulum (ER) stress and oxidative stress was attenuated by melatonin supplementation. In conclusion, these findings demonstrate a protective role of melatonin against Aß peptide accumulation, ER stress and oxidative stress in the AlCl3 -treated AD model. Hence, the melatonin supplement might be an alternative way to alleviate the development of AD.

The induction of Neuron-Glial2 (NG2) expressing cells in methamphetamine toxicity-induced neuroinflammation in rat brain are averted by melatonin.

Neuron-Glial2 (NG2) expressing cells are described as the oligodendrocyte precursor cells in the brain. This study aimed to investigate the possible involvement of NG2 cells under the methamphetamine (METH)-induced neurotoxicity and neuroprotective capacity of melatonin. The results showed that the levels of NG2 in rat brain gradually increase from postnatal day 0 to postnatal day 8 and then the lower levels of NG2 are shown in adults. In adult rats, the levels of NG2 and COX-2 in the brain were significantly increased in lipopolysaccharide treatment. Pretreatment of 10 mg/kg melatonin prior to treating with METH was able to reduce an increase in the levels of NG2 and activation in astrocyte and microglia. These findings would extend the contribution of NG2 expressing cells in the adult brain during pathological conditions such as neuroinflammation.

Melatonin prevents calcineurin-activated the nuclear translocation of nuclear factor of activated T-cells in human neuroblastoma SH-SY5Y cells undergoing hydrogen peroxide-induced cell death.

The interaction between the activation of protein phosphatase, calcineurin (CaN), and the dephosphorylation and nuclear translocation of nuclear factor of activated T-cells (NFAT), a transcriptional factor in the immune system, has attracted interest as a key factor responsible for the cell death process. In this study, the effects of melatonin on the interaction between CaN and NFAT signaling during oxidative stress-induced cell death were investigated. Human neuroblastoma SH-SY5Y cells were treated with the non-radical reactive oxygen species hydrogen peroxide (H2O2). Cells were treated with 200 µM H2O2 for the indicated time. Some H2O2-treated cells were pretreated with melatonin for 1 h. Control cells were treated with the same concentration of ethanol used to dilute melatonin. H2O2-induced cell death promoted increases in reactive oxygen species (ROS) production and the nuclear translocation of NFAT, which were related to increased levels the active, cleaved form of CaN (32.5 kDa). In addition, pretreatment of H2O2-treated cells with melatonin decreased cell death, ROS production, the levels of the active-cleaved form of CaN and the nuclear translocation of NFAT. Based on these findings, melatonin may exert its neuroprotective effects on oxidative damage-induced cell death by inhibiting CaN-activated the nuclear translocation of NFAT.

Protection against cell death and sustained tyrosine hydroxylase phosphorylation in hydrogen peroxide- and MPP-treated human neuroblastoma cells with melatonin.

Neuroprotective effects of melatonin against oxidative stress-induced neuronal cell degeneration in human SH-SY5Y neuroblastoma cells were investigated in this report. The results demonstrate that exogenous administration of H(2)O(2) and 1-methyl, 4-phenyl, pyridinium ion (MPP(+)) significantly decreased cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker was able to abolish the toxic effects of MPP(+) but not H(2)O(2) in reduction of cell viability. Conversely, melatonin reversed the toxic effects of H(2)O(2) and MPP(+) on cell viability. In addition, the reduction of phosphorylation of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and phosphorylation of cyclic AMP responsive element-binding protein by H(2)O(2) and MPP(+) was also diminished by melatonin. These results demonstrate some effective roles of melatonin on neuroprotection and its action on the modulation of tyrosine hydroxylase phosphorylation.