Neuroprotective effects of melatonin on amphetamine-induced dopaminergic fiber degeneration in the hippocampus of postnatal rats.

Chronic amphetamine (AMPH) abuse leads to damage of the hippocampus, the brain area associated with learning and memory process. Previous results have shown that AMPH-induced dopamine neurotransmitter release, reactive oxygen species formation, and degenerative protein aggregation lead to neuronal death. Melatonin, a powerful antioxidant, plays a role as a neuroprotective agent. The objective of this study was to investigate whether the protective effect of melatonin on AMPH-induced hippocampal damage in the postnatal rat acts through the dopaminergic pathway. Four-day-old postnatal rats were subcutaneously injected with 5-10 mg/kg AMPH and pretreated with 10 mg/kg melatonin prior to AMPH exposure for seven days. The results showed that melatonin decreased the AMPH-induced hippocampal neuronal degeneration in the dentate gyrus, CA1, and CA3. Melatonin attenuated the reduction in the expression of hippocampal synaptophysin, PSD-95, α-synuclein, and N-methyl-D-aspartate (NMDA) receptor protein and mRNA caused by AMPH. Melatonin attenuated the AMPH-induced reduction in dopamine transporter (DAT) protein expression in the hippocampus and the reduction in mRNA expression in the ventral tegmental area (VTA). Immunofluorescence demonstrated that melatonin not only prevented the AMPH-induced loss of DAT and NMDA receptor but also prevented AMPH-induced α-synuclein overexpression in the dentate gyrus, CA1, and CA3. Melatonin decreased the AMPH-induced reduction in the protein and mRNA of the NMDA receptor downstream signaling molecule, calcium/calmodulin-dependent protein kinase II (CaMKII), and the melatonin receptors (MT1 and MT2). This study showed that melatonin prevented AMPH-induced toxicity in the hippocampus of postnatal rats possibly via its antioxidative effect and mitochondrial protection.

Effects of melatonin on severe crush spinal cord injury-induced reactive astrocyte and scar formation.

The present work aimed at analyzing the effects of melatonin on scar formation after spinal cord injury (SCI). Upregulation of reactive astrocyte under SCI pathological conditions has been presented in several studies. It has been proved that the crucial factor in triggering this upregulation is proinflammatory cytokines. Moreover, scar formation is an important barrier to axonal regeneration through the lesion area. Melatonin plays an important role in reducing inflammation, but its effects on scar formation in the injured spinal cord remain unknown. Hence, we used the model of severe crush injury in mice to investigate the effects of melatonin on scar formation. Mice were randomly separated into four groups; SCI, SCI+Melatonin 1 (single dose), SCI+Melatonin 14 (14 daily doses), and control. Melatonin was administered by intraperitoneal injection (10 mg/kg) after injury. Immunohistochemical analysis, Western blot, and behavioral evaluation were used to explore the effects of melatonin after SCI for 14 days. The melatonin-treated mice presented higher expression of neuronal markers (P < 0.001). Remarkably, the inflammatory response appeared to be greatly reduced in the SCI+Melatonin 14 group (P < 0.001), which also displayed less scar formation (P < 0.05). These findings suggest that melatonin inhibits scar formation by acting on inflammatory cytokines after SCI. Overall, our results suggest that melatonin is a promising treatment strategy after SCI that deserves further investigation. © 2016 Wiley Periodicals, Inc.

Anxiety-like behaviour and c-fos expression in rats that inhaled vetiver essential oil.

Vetiver essential oil (VEO) has been used in aromatherapy for relaxation. This study aimed to investigate the effects of VEO on an anxiety-related behavioural model (the elevated plus-maze, EPM) and immediate-early gene c-fos in amygdala, known to be involved in anxiety. Male Wistar rats were administered diazepam (1 mg/kg i.p.) for 30 min or inhalated with VEO (1%, 2.5% or 5% w/w) for 7 min prior to exposure to the EPM. Then, the effects of 2.5% VEO, the anxiolytic dose, on c-fos expression in amygdala were investigated. The rats given either 2.5% VEO or diazepam exhibited an anxiolytic-like profile in the EPM. VEO and diazepam significantly increased c-fos expression in the lateral division of the central amygdaloid nucleus (CeL). Therefore, the anxiolytic properties of VEO might be associated with altering neuronal activation in CeL. However, future studies are needed to investigate the precise mechanism of action of VEO.

Development of Clock Genes Expression in Rat Hippocampus.

BACKGROUND: The circadian rhythms in the suprachiasmatic nucleus (SCN), a central clock, are generated by autoregulatory network composed ofclock genes that encode transcriptionalfactors. There is a gradual development ofclock gene expression in the SCN during ontogenesis. Moreover clock genes are expressed in the adult hippocampus with circadian fashion. OBJECTIVE: It is of interest to examine daily profiles ofthe clock gene mRNA and protein expressions in rat hippocampus during development. MATERIAL AND METHOD: Daily profiles ofthree clock genes (Per1, Per2, and Bmal1) mRNA, and their protein expressions were analyzed in the rat hippocampus ofpups at postnatal (P) day 4 and 8 (P4 and P8), pre-weaning stage (P16), early pubertal stage (P32), and adult (P60) by real-time PCR and immunohistochemistry. RESULTS: The entire studied clock gene mRNAs and proteins did not exhibit circadian rhythm in early postnatal P4-P16. Rhythmic expression of Per1 and Per2 mRNA started at P32, whereas Bmal1 began at adult. However, their proteins showed circadian expression together at adult. CONCLUSION: The present study suggests that rat hippocampal molecular clock works gradually develop after birth and slower than that in the central clock SCN. It was possible that ontogenetic development of clock gene in hippocampus was waitingfor central clocksynchronization.

Neuropeptide Y in the adult and fetal human pineal gland.

Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally.

Effect of amphetamine on the clock gene expression in rat striatum.

Drug addicts have severe disruptions in many physiological and behavioral rhythms, such as the sleep/wake cycle. Interestingly, amphetamine, a psychostimulant, is able to alter many circadian patterns, which are independent of the master biological clock located in the suprachiasmatic nucleus. To increase our understanding of the circadian regulation of amphetamine on clock gene expression, rats received subcutaneous injections of d-amphetamine and the clock gene mRNA levels were analyzed using real-time PCR to obtain a daily profile. In the striatum, acute injection of d-amphetamine did not alter Period (Per)1, Per2 and Reverse erythroblastosis virus α (Rev-erbα) expressions. Chronic administration shifted the phase of Per1 and Per2 expressions from a nocturnal to diurnal pattern and advance shifted the peak of Rev-erbα in d-amphetamine-treated animals. In contrast, the rhythm of Brain and muscle Arnt-like protein-1 (Bmal1) was shifted from a diurnal to a nocturnal pattern by both acute and chronic treatments. These results demonstrated that chronic d-amphetamine treatment altered the expression of clock genes in the striatum. This might further influence the expression of related gene within the striatum and lead to behavioral and physiological changes which are associated to drug addiction.

Calpastatin reduces calpain and caspase activation in methamphetamine-induced toxicity in human neuroblastoma SH-SY5Y cultured cells.

Methamphetamine (METH) is an abused psychostimulant drug that can cause neurotoxicity to dopaminergic cells. It has been demonstrated that METH can induce caspase- and calpain-dependent death cascades. The purpose of the present study was to investigate the functional role of calpastatin, a specific endogenous calpain inhibitor protein, on caspase and calpain activation in METH-induced degeneration in neuroblastoma SH-SY5Y cell cultures. In this study, we found that METH significantly decreased cell viability, tyrosine hydroxylase phosphorylation and calpastatin levels. Supplementation of cells with exogenous calpastatin was able to reverse the toxic effect of METH on reduction in cell viability and tyrosine hydroxylase phosphorylation. METH also significantly increased calpain levels, the formation of calpain-specific breakdown products and cleaved caspase-3 levels; once again, these effects were diminished by pretreating the cells with calpastatin. These data suggest the contribution of calpastatin as a potential regulatory factor for calpain- and caspase-dependent death processes.

The expression of Per1 and Aa-nat genes in the pineal gland of postnatal rats.

BACKGROUND: The circadian rhythm of melatonin synthesis is controlled by the master clock, suprachiasmatic nucleus (SCN). The level of melatonin changes throughout the aging process. The SCN's rhythm is driven by autoregulatory feedback loop composed of a set of clock genes families and their corresponding proteins. The Period (Per1), one of clock gene develops gradually during postnatal ontogenesis in the rat SCN and is also expressed in the pineal gland. OBJECTIVE: It is of interest to study the relationship between the postnatal development of Per1 and Aa-nat, genes that produce the rate-limiting enzyme in melatonin synthesis, in the pineal. MATERIAL AND METHOD: Daily profiles of mRNA expression of Per1 and Aa-nat were analyzed in the pineal gland of pups at postnatal ages 4 (P4), P8, P16 and P32, at puberty age of 6 weeks; and in 8 week-old adult rats by real-time PCR. RESULTS: As early as P4, Per1 and Aa-nat mRNAs were expressed and existed at relatively high levels during the nighttime. They gradually increased until puberty and decreased at 8 weeks of age. Additionally, the nocturnal changes of Per1 and Aa-nat mRNA levels in the rat pineal gland from P4 to adults were strongly correlated at r = 0.97 (p < 0.01). CONCLUSION: The present data indicate that there is a close relationship between the expression pattern of Per1 and that of melatonin synthesis during the development of postnatal rats.

Melatonin reduces the expression of alpha-synuclein in the dopamine containing neuronal regions of amphetamine-treated postnatal rats.

Alpha-synuclein (α-syn) is a neuronal protein that is involved in various degenerative disorders such as Parkinson's disease. It is found in the presynaptic terminals and perinuclear zones of many brain regions. Amphetamine (AMPH), a psychostimulant drug abused progressively more commonly in recent years, has been known to induce neurotoxicity in the central dopaminergic pathway, which is associated with increased oxidative stress. Recently, AMPH has been shown to significantly increase the level of α-syn in dopaminergic neuroblastoma cell cultures. Melatonin is recognized as an antioxidant for the nervous system. This study tested whether melatonin can attenuate the effect of AMPH on the expression of α-syn in the dopaminergic pathway of the neonatal rat. Four-day old postnatal rats (P4) were injected subcutaneously with either AMPH (increasing dose, 5-10 mg/kg daily) alone or AMPH with melatonin (2 mg/kg) daily at 10:00 AM for 7 consecutive days. As determined using Western blot, the level of α-syn was significantly increased in the substantia nigra, dorsal striatum, nucleus accumbens, and prefrontal cortex of the AMPH-treated group, while melatonin treatment either prior to AMPH or alone decreased the accumulation of the protein to 77%, 96%, 78%, and 77% of the control value, respectively. Furthermore, an immunofluorescent study showed that the α-syn-immunoreactivity increased noticeably in the nuclei of cell bodies and nerve terminals of the AMPH-treated group. Again, melatonin lowered this immunoreactivity. These results indicate that melatonin has a direct or indirect effect in reducing the expression of α-syn in the postnatal rat. The exact mechanism of this mitigation should be further investigated.

Clinical identification for the use of light touch cues with a cane in gait rehabilitation poststroke.

OBJECTIVES: To determine clinical characteristics that can be used to identify patients with stroke who can perform light touch contact with a cane during walking, and to determine whether these patients benefit from TC. METHODS: A prediction and randomized experimental study was conducted of 62 patients (mean onset time, 43.8 days) who were 59.4 ± 11.2 years. There were 3 conditions of cane usage: force contact (FC), touch contact (TC), and no contact (NC). Clinical characteristics included age, stroke onset duration, gait speed, and Chedoke-McMaster and Fugl-Meyer (FM) Assessment scores. We studied trunk acceleration and activation of tensor fascia latae (TFL), and vastus medialis (VM) muscles during walking using 3 cane conditions. RESULTS: Out of 62 patients, 36 were able to perform TC during walking. These patients benefited from TC as demonstrated by higher trunk stability (compared to NC) and larger paretic VM and TFL activations (compared to FC). FM scale was the only variable that differed between patients who could perform TC and those who could not (P < .00). Analysis of receiver operating characteristics (ROC) revealed that FM scores for balance and lower extremity motor domains (area under ROC = 0.93 and 0.96, respectively) are able to predict the TC group with high accuracy. Calculations using cutoff scores for balance (6.5) or lower extremity (14.5) FM subscales correctly predicted patients who benefited from TC 89% of the time. CONCLUSION: Balance and lower extremity FM subscales can be used to identify suitable candidates among patients with stroke for implementation of TC in gait rehabilitation.

A noradrenergic sensitive endogenous clock is present in the rat pineal gland.

The aim of this study was to examine the occurrence of endogenous oscillations of Per1, Per2, Bmal1 and Rev-erbα genes in rat pineal explants and to investigate their regulation by adrenergic ligands. Our results show a significant and sustained rhythm of Per2,Bmal1 and Rev-erbα gene expression for up to 48 h in cultured pineal gland with a pattern similar to that observed in vivo. By contrast, the rhythms of Per1 and Aa-nat, the rate-limiting enzyme for melatonin synthesis, were strongly attenuated after 24 h in culture. Addition of the exogenous adrenergic agonist isoproterenol on cultured pineal glands induced a short-term increase in mRNA levels of Per1 and Aa-nat, but not those of Per2,Bmal1 and Rev-erbα. This study demonstrates that the rat pineal gland hosts a circadian oscillator as evidenced by the sustained, noradrenergic-independent, endogenous oscillations of Per2, Bmal1 and Rev-erbα mRNA levels in cultured tissues. Only expression of Per1 was stimulated by adrenergic ligands suggesting that, in vivo, the adrenergic input could synchronize the pineal clock by acting selectively on Per1.

Melatonin increases proliferation of cultured neural stem cells obtained from adult mouse subventricular zone.

Melatonin, a circadian rhythm-promoting molecule secreted mainly by the pineal gland, has a variety of biological functions and neuroprotective effects including control of sleep-wake cycle, seasonal reproduction, and body temperature as well as preventing neuronal cell death induced by neurotoxic substances. Melatonin also modulates neural stem cell (NSC) function including proliferation and differentiation in embryonic brain tissue. However, the involvement of melatonin in adult neurogenesis is still not clear. Here, we report that precursor cells from adult mouse subventricular zone (SVZ) of the lateral ventricle, the main neurogenic area of the adult brain, express melatonin receptors. In addition, precursor cells derived from this area treated with melatonin exhibited increased proliferative activity. However, when cells were treated with luzindole, a competitive inhibitor of melatonin receptors, or pertussis toxin, an uncoupler of Gi from adenylate cyclase, melatonin-induced proliferation was reduced. Under these conditions, melatonin induced the differentiation of precursor cells to neuronal cells without an upregulation of the number of glia cells. Because stem cell replacement is thought to play an important therapeutic role in neurodegenerative diseases, melatonin might be beneficial for stimulating endogenous neural stem 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.

Repeated restraint stress and corticosterone injections during late pregnancy alter GAP-43 expression in the hippocampus and prefrontal cortex of rat pups.

In the offspring of prenatal stress animals, overactivity and impaired negative feedback regulation of the hypothalamic-pituitary-adrenal axis are consistent finding. However, little was known about how prenatal stress can permanently alter developmental trajectories of pup's brain. Growth-associated protein-43 (GAP-43) is a presynaptic membrane phosphoprotein whose expression increases during developmental events such as axonal outgrowth or remodeling and synaptogenesis. Phosphorylation of GAP-43 by protein kinase C was correlated with enhanced axonal growth and transmitter release. In adult animals, increase of GAP-43 correlated with monoaminergic deficit in neuropsychiatric disorders. The present study examines the effects of repeated maternal restraint stress on the level of GAP-43 in the brain of rat pups. The results showed that prenatal stress significantly increased GAP-43 level in the PFC of rat pup during PND 7-14 as compared to control but not significant difference when observed at PND 21. Increased GAP-43 expression was also observed in the pup's hippocampus during the same postnatal periods. However, when observed at PND 60, pups born from stressed mother showed a significant lower (p<0.001) GAP-43 expression as compare with control group. These changes indicate the direct effect of corticosteroid hormone, since repeated maternal injection with corticosterone (CORT, 40 mg/kg) during GD 14-21 also gave the same results. PND 7-14 is the peak period of synaptogenesis in these brain areas and abnormal axon sprouting and reorganization may lead to a defect in synaptic pruning at later stage of life. The results suggested that maternal stress is harmful to the developing brain and upregulation of GAP-43 indicated a protective mechanism against the toxicity of maternal stress hormone. Prenatal stress alter the normal developmental trajectories in the pup's brain may underlies the mechanism link between early life stress and neuropsychopathology in later life.

Light touch cue through a cane improves pelvic stability during walking in stroke.

OBJECTIVE: To examine the effect of a light touch cue provided through a cane on mediolateral (ML) pelvic stability during walking in subjects poststroke. DESIGN: Crossover trial examining ML pelvic stability during walking using a cane with the force contact and touch contact methods. SETTING: Physical therapy clinic, tertiary care center. PARTICIPANTS: Subacute patients (N=40) with stroke with a mean age of 59.6 years and mean stroke duration of 46.8 days. The average gait speed with a cane was .13 m/s (.05-.29 m/s). INTERVENTION: Using a cane with the force contact and touch contact methods during walking. MAIN OUTCOME MEASURES: ML pelvic stability as measured by averaged peak-to-peak pelvic acceleration, muscle activation of bilateral tensor fascia latae (TFL), semitendinosus (ST), and vastus medialis (VM) using an electromyography system, and vertical cane force. RESULTS: The average amount of cane force during touch contact and force contact cane use conditions was 2.3N and 49.3N, respectively. A light touch cue through a cane was required only when the paretic leg accepted the body weight, and this cue can provide ML pelvic stability (.16 g of average pelvic acceleration) during walking to the same degree as the force contact method of cane use. However, significant increases in single-limb support duration with higher activations of TFL, VM, and ST muscles on the paretic leg were found during the paretic stance phase when using a cane in the touch contact fashion (P<.05). CONCLUSIONS: A light touch cue can be provided during walking through the use of a cane. This augmented somatosensory information provides lateral stability during walking for subjects with stroke by facilitating the activations of weight-bearing muscles on the paretic leg during the stance phase.

Endogenous rhythmicity of Bmal1 and Rev-erb alpha in the hamster pineal gland is not driven by norepinephrine.

Pineal melatonin is synthesized with daily and seasonal rhythms following the hypothalamic clock-driven release of norepinephrine (NE). The pineal gland of rats and mice, like the biological clock, expresses a number of clock genes. However, the role of pineal clock elements in pineal physiology is still unknown. We examined the expression and regulation of several clock genes (Per1, Cry2, Bmal1 and Rev-erb alpha) under different lighting conditions or following adrenergic treatments in the Syrian hamster, a seasonal rodent. We found that Per1 and Cry2 genes were similarly regulated by the nocturnal release of NE: levels of Per1 and Cry2 mRNA displayed a nocturnal increase that was maintained after 2 days in constant darkness (DD) but abolished after 2 days under constant light (LL), a condition that suppresses endogenous NE release, or after an early night administration of the adrenergic antagonist propranolol. In contrast, Bmal1 and Rev-erb alpha exhibited a different pattern of expression and regulation. mRNA levels of both clock genes displayed a marked daily variation, maintained in DD, with higher values at midday for Bmal1 and at day/night transition for Rev-erb alpha. Remarkably, the daily variation of both Bmal1 and Rev-erb alpha mRNA was maintained in LL conditions and was not affected by propranolol. This study confirms the daily regulation of Per1 and Cry2 gene expression by NE in the pineal gland of rodents and shows for the first time that a second set of clock genes, Bmal1 and Rev-erb alpha are expressed with a circadian rhythm independent of the hypothalamic clock-driven noradrenergic signal.

Melatonin attenuates methamphetamine-induced reduction of tyrosine hydroxylase, synaptophysin and growth-associated protein-43 levels in the neonatal rat brain.

Methamphetamine (METH) is a most commonly abused drug which damages nerve terminals by causing formation of reactive oxygen species (ROS), apoptosis, and finally neuronal damage. Fetal exposure to neurotoxic METH causes significant behavioral effects. The developing fetus is substantially deficient in most antioxidative enzymes, and may therefore be at high risk from both endogenous and drug-enhanced oxidative stress. Little is known about the effects of METH on vesicular proteins such as synaptophysin and growth-associated protein 43 (GAP-43) in the immature brain. The present study attempted to investigate the effects of METH-induced neurotoxicity in the dopaminergic system of the neonatal rat brain. Neonatal rats were subcutaneously exposed to 5-10mg/kg METH daily from postnatal day 4-10 for 7 consecutive days. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the dorsal striatum, prefrontal cortex, nucleus accumbens and substantia nigra, synaptophysin levels decreased in the striatum and prefrontal cortex and growth-associated protein-43 (GAP-43) levels significantly decreased in the nucleus accumbens of neonatal rats. Pretreatment with 2mg/kg melatonin 30 min prior to METH administration prevented METH-induced reduction in tyrosine hydroxylase, synaptophysin and growth-associated protein-43 protein levels in different brain regions. These results suggest that melatonin provides a protective effect against METH-induced nerve terminal degeneration in the immature rat brain probably via its antioxidant properties.

Melatonin reduces induction of Bax, caspase and cell death in methamphetamine-treated human neuroblastoma SH-SY5Y cultured cells.

Several studies demonstrated that methamphetamine (MA)-treated human neuroblastoma cells exhibit increased oxidative stress, which regulates intracellular signaling cascades leading to cell death. Melatonin has a potential as a direct free radical scavenger and protects against cell death caused by MA. The objective of this study was to investigate the neuroprotective properties of melatonin on MA-induced induction of death signaling cascade and neuronal cell degeneration in human neuroblastoma SH-SY5Y cultured cells. The results of the present study demonstrate that MA significantly reduced cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker, and melatonin reversed the toxic effect of MA in reducing cell viability. Induction of Bax, Bcl-2 and cleaved caspase-3 protein levels were observed in SH-SY5Y cultured cells treated with MA, whereas the induction of Bax and cleaved caspase-3 was diminished by melatonin. Visualization of the induction of Bax using immunofluorescence but a reduction in mitochondrial sites using red-fluorescent mitochondria-staining dye was more obviously apparent in MA-treated cells than in untreated control cells and, again, this effect was abolished by melatonin. These findings demonstrate important roles of Bax and caspase in death signaling cascade, and the protective effects of melatonin in MA-treated SH-SY5Y cells.

Melatonin inhibits amphetamine-induced increase in alpha-synuclein and decrease in phosphorylated tyrosine hydroxylase in SK-N-SH cells.

alpha-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegeneration disorders. Understanding alpha-synuclein function in dopaminergic cells could add to our knowledge of this key protein which is implicated in Parkinson's disease. Chronic or intermittent amphetamine (AMPH) abuse may create temporary or permanent disturbances in the dopaminergic system of the brain that may predispose individuals to Parkinsonism. Our previous studies showed that neurotoxicity induced by AMPH was mediated by enhanced oxidative stress and these effects were abolished by melatonin, a main secretory product of pineal gland. The present study was conducted to investigate the effect of AMPH on alpha-synuclein in regulating tyrosine hydroxylase (TH), a rate limiting enzyme for dopamine synthesis, in cultured human dopaminergic SK-N-SH cells. Of these, phosphorylation of Ser40 (pSer40) contributes significantly to TH activation and dopamine synthesis. Our data indicated that AMPH significantly increased the level of alpha-synuclein to 183% of the control value while reducing the levels of phosphorylated TH (TH-pSer40) enzyme and mitochondrial complex I to 78 and 52.9% of the control values, respectively and these effects were attenuated by melatonin. Further studies are needed to explore the mechanism by which alpha-synuclein contributes to TH-pSer40 dephosphorylation and the mechanism by which melatonin contributes to this interaction.

Melatonin protects SK-N-SH neuroblastoma cells from amphetamine-induced neurotoxicity.

Several hypotheses regarding the mechanism underlying amphetamine-induced neurotoxicity have been proposed. One of them is based on the observation of free radical formation and oxidative stress produced by auto-oxidation of dopamine (DA). The formation of DA-related reactive oxygen species (ROS) such as superoxide and hydroxyl radicals appears to play an important role in amphetamine-induced neurotoxicity. Melatonin, the main secretory product of pineal gland, is well known for its protective effects that are currently attributed mainly to its radical scavenging and antioxidant properties. The present study was conducted to investigate the protective effects of melatonin on d-amphetamine (AMPH)-induced neurotoxicity in cultured human dopaminergic neuroblastoma SK-N-SH cells. Our data indicate that AMPH significantly reduces cell viability, induces oxidative stress (enhances ROS production and malondialdehyde levels), up-regulates alpha-synuclein expression and decreases intracellular ATP levels. However, pretreatment of SK-N-SH cells with melatonin prevents AMPH-induced loss of cell viability and induction of oxidative stress, while reducing alpha-synuclein expression and increasing ATP production. These results suggest that the antioxidant properties of melatonin may provide a protective mechanism against AMPH-induced neuronal degeneration.