The neuroprotective effect of melatonin against the induction of c-Jun phosphorylation by 6-hydroxydopamine on SK-N-SH cells

Melatonin is synthesized mainly in pineal gland. It has been suggested that melatonin has proven antioxidant effects and protective effects against neuronal cell degeneration. There are several studies indicating that c-Jun-N-terminal kinase pathways might be involved in neuronal cell death. In this study, the effects of melatonin on 6-hydroxydopamine (6-OHDA)-treated cultured SK-N-SH cells were investigated. The results showed that 6-OHDA significantly decreased cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, and melatonin was able to reverse the toxic effects of 6-OHDA on cell viability. In addition, induction of c-Jun phosphorylation by 6-OHDA was diminished by melatonin. These results demonstrate some protective properties of melatonin against neuronal cell degeneration and its action on the inhibition of c-Jun-N terminal kinase signaling cascade.     

The potentiating effect of calcitonin gene-related peptide on transient receptor potential vanilloid-1 activity and the electrophysiological responses of rat trigeminal neurons to nociceptive stimuli

Growing evidence suggests that calcitonin gene-related peptide (CGRP) participates in trigeminal nociceptive responses. However, the role of CGRP in sensitization or desensitization of nociceptive transduction remains poorly understood. In this study, we sought to further investigate the CGRP-induced up-regulation of transient receptor potential vanilloid-1 (TRPV1) and the responses of trigeminal neurons to nociceptive stimuli. Rat trigeminal ganglion (TG) organ cultures and isolated trigeminal neurons were incubated with CGRP. An increase in TRPV1 levels was observed in CGRP-incubated TG organ cultures. CGRP potentiated capsaicin-induced increase in phosphorylated CaMKII levels in the TG organ cultures. The incubation of the trigeminal neurons with CGRP significantly increased the inward currents in response to capsaicin challenge, and this effect was inhibited by co-incubation with the CGRP receptor antagonist, BIBN4068BS or the inhibitor of protein kinase A, H-89. These findings reveal that CGRP acting on trigeminal neurons may play a significant role in facilitating cellular events that contribute to the peripheral sensitization of the TG in nociceptive transmission.     

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

Abstract:  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 H₂O₂ 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 H₂O₂ in reduction of cell viability. Conversely, melatonin reversed the toxic effects of H₂O₂ 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 H₂O₂ 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.     

Melatonin inhibits MPP+-induced caspase-mediated death pathway and DNA fragmentation factor-45 cleavage in SK-N-SH cultured cells

Neurodegenerative diseases such as Parkinson's disease are illnesses associated with high morbidity and mortality with few, or no effective, options available for their treatment. In addition, the direct cause of selective dopaminergic cell loss in Parkinson's disease has not been clearly understood. Taken together, several studies have demonstrated that melatonin has a neuroprotective effect both in vivo and in vitro. Accordingly, the effects of melatonin on 1-methyl, 4-phenyl, pyridinium ion (MPP(+))-treated cultured human neuroblastoma SK-N-SH cell lines were investigated in the present study. The results showed that MPP(+) significantly decreased cell viability. By contrast, an induction of phosphorylation of c-Jun, activation of caspase-3 enzyme activity, cleavage of DNA fragmentation factors 45 and DNA fragmentation were observed in MPP(+)-treated cells. These changes were diminished by melatonin. These results demonstrate the cellular mechanisms of neuronal cell degeneration induced via c-Jun-N-terminal kinases and caspase-dependent signaling, and the potential role of melatonin on protection of neuronal cell death induced by this neurotoxin.     

The presence of melatonin receptors and inhibitory effect of melatonin on hydrogen peroxide-induced endothelial nitric oxide synthase expression in bovine cerebral blood vessels

Melatonin plays a key role in a variety of important physiological functions including influencing cerebral blood vessels. Therefore, in the present study, we have identified the existence of melatonin receptors and test the effect of melatonin on hydrogen peroxide-induced endothelial nitric oxide synthase (eNOS) expression in bovine cerebral arteries. The results indicate that mt1A melatonin receptor mRNA is expressed in bovine cerebral arteries. The relative levels of mt1A melatonin receptor mRNA expression in anterior, posterior, middle and vertebral cerebral arteries were compared. The data show the highest and lowest levels of mRNA expressions in the middle and vertebral cerebral arteries, respectively. The maximal number (B(max)) of different types of melatonin receptors in various regions of cerebral arteries were identified and further characterized by using the selective 2-[(125)I] iodomelatonin binding assay. Saturation studies revealed that the binding represented a single site of high affinity binding for the melatonin receptor with the highest and lowest binding capacities in the middle and vertebral arteries, respectively. In order to elaborate the functional significance of melatonin in cerebral blood vessels, hydrogen peroxide- induced induction in eNOS protein level and phosphorylation of calcium/calmodulain-dependent protein kinase II (phospho-CaMKII) were demonstrated in the bovine isolated cerebral arteries with these effect being abolished by melatonin. This is the first evidence showing expression of mt1A melatonin receptor in the bovine cerebral arteries. However, further studies are necessary to delineate the role of melatonin and its receptors in regulating physiology of the cerebral vessels.     

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

Abstract:  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.     

Deferoxamine attenuates iron-induced oxidative stress and prevents mitochondrial aggregation and alpha-synuclein translocation in SK-N-SH cells in culture

One of the defining characteristics of neurodegenerative diseases, including Parkinson's disease, is an abnormal accumulation of iron in the affected brain areas. By using SK-N-SH, a dopaminergic cell line, we have found that iron (100-250 microM FeSO(4)) decreased cell viability, increased lipid peroxidation, and the said effects were blocked by deferoxamine (DFO: 10 microM). Furthermore, DFO, in the absence of iron, enhanced the level of adenosine triphosphate (ATP), but caused chromatin condensation and cell death. Morphological studies revealed that iron (50-100 microM) altered mitochondrial morphology, disrupted nuclear membrane, and translocated alpha-synuclein from perinuclear region into the disrupted nucleus. The results of these studies suggest that DFO is able to block and attenuate iron-mediated oxidative stress. However, in the absence of excess iron, DFO itself may have deleterious effects on the morphology and hence integrity of dopaminergic neurons.     

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, Plp^4e/‐. During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial‐selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp^4e/‐ 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.     

How should left atrial size be reported? Comparative assessment with use of multiple echocardiographic methods

Background

Determination of left atrial (LA) size is important in clinical decision-making. The LA anteroposterior dimension (APD) has been routinely reported as LA size assessment. Early studies indicated that the APD may have limited accuracy in quantification. Conventional 3-dimensional reconstruction (C3DR) of the LA has been validated. However, its process is time-consuming and not applicable for daily practice. To explore an accurate and practical approach, we compared different echocardiographic measurements with C3DR in 141 patients with different LA sizes.

Methods and results

LA size was measured with (1) the cubic equation with APD (Cub); (2) the ellipsoidal formula (Ellp); (3) biplane modified Simpson rule (biplane); and (4) simplified 3-dimensional reconstruction from 3 standard apical views with B spline interpolation (S3VR). All four methods were compared with C3DR. S3VR and biplane methods provided a close agreement to C3DR (y = 0.94x + 3.6, r = 0.95, SEE = 7.6 mL, mean difference = −1.3% for S3VR; y = 0.87x + 2.9, r = 0.91, SEE = 9.0 mL, mean difference = −9.4% for biplane). The Cub and Ellp calculations were less accurate, with significant volume underestimation (P < .001).

Conclusions

LA single dimension is not accurate for LA size measurement. Among four different methods of LA size measurement, biplane and S3VR provide the closest agreement to C3DR. The biplane, which is readily applicable with current echocardiographic equipment, should be routinely applied in clinical practice.     

The effects of adrenergic agonists on c-fos, jun-B and nitric oxide synthase-like immunoreactivities in cultured rat pinealocytes

Pineal melatonin synthesis is enhanced by the activation of adrenergic and opioid receptors. The precise mechanism of how signal transduction is affected by the activation of these receptors, leading to an increase in melatonin synthesis, is not clear. An attempt has been made to investigate the effects of the activation of these two types of receptors that might lead to the induction of immediate early genes (IEGs) and nitric oxide synthase (NOS) expressions. The present study shows that the stimulation of cultured pinealocytes by 1 microM epinephrine (an alpha- and beta-adrenergic agonist) for 2 h increased the number of c-fos immunoreactive (IR) cells, and that this stimulatory effect was abolished by adding 10 microM prazosin (an alpha-adrenergic antagonist) to the culture medium. No significant change of c-fos-IR cells was found when cells were treated with either 1 microM isoproterenol (a beta-adrenergic agonist) or 100 microM morphine (an opioid agonist). Neither epinephrine (1 microM), isoproterenol (1 microM) nor morphine (100 microM) altered jun-B-IR cells in the culture. With regard to NOS, the number of IR cells was slightly increased after 2 h of incubation by 10 microM isoproterenol, whereas morphine (100 microM) and epinephrine (10 microM) did not change the number of IR cells. The results of this experiment have demonstrated that c-fos expression is induced by alpha-adrenergic but not beta-adrenergic agonists, nor by opioid agonists. Neither alpha-, beta-adrenergic nor opioid agonists had any significant effect on jun-B, whereas NOS was slightly increased by isoproterenol.