Lennard P. Niles

Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT 1 receptor with neuronal and glial markers

BackgroundIn order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs.ResultsRT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (β-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours.ConclusionsThe phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT1 receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinsons disease.

Novel targets for valproic acid: up-regulation of melatonin receptors and neurotrophic factors in C6 glioma cells

Valproic acid (VPA) is a potent anti‐epileptic and effective mood stabilizer. It is known that VPA enhances central GABAergic activity and activates the mitogen‐activated protein kinase–extracellular signal‐regulated kinase (MAPK–ERK) pathway. It can also inhibit various isoforms of the enzyme, histone deacetylase (HDAC), which is associated with modulation of gene transcription. Recent in vivo studies indicate a neuroprotective role for VPA, which has been found to up‐regulate the expression of brain‐derived neurotrophic factor (BDNF) in the rat brain. Given the interaction between the pineal hormone, melatonin, and GABAergic systems in the central nervous system, the effects of VPA on the expression of the mammalian melatonin receptor subtypes, MT1 and MT2, were examined in rat C6 glioma cells. The effects of VPA on the expression of glial cell line‐derived neurotrophic factor (GDNF) and BDNF were also examined. RT‐PCR studies revealed a significant induction of melatonin MT1 receptor mRNA in C6 cells following treatment with 3 or 5 mm VPA for 24 h or 5 mm VPA for 48 h. Western analysis and immunocytochemical detection confirmed that the VPA‐induced increase in MT1 mRNA results in up‐regulation of MT1 protein expression. Blockade of the MAPK–ERK pathway by PD98059 enhanced the effect of VPA on MT1 expression, suggesting a negative role for this pathway in MT1 receptor regulation. In addition, significant increases in BDNF, GDNF and HDAC mRNA expression were observed after treatment with VPA for 24 or 48 h. Taken together, the present findings suggest that the neuroprotective properties of VPA involve modulation of neurotrophic factors and receptors for melatonin, which is also thought to play a role in neuroprotection. Moreover, the foregoing suggests that combinations of VPA and melatonin could provide novel therapeutic strategies in neurological and psychiatric disorders.

Human malignant melanoma cells express high-affinity receptors for melatonin: antiproliferative effects of melatonin and 6-chloromelatonin

In order to explore the potential oncostatic properties of the pineal hormone, melatonin, we have investigated its binding characteristics and functional effects in a human malignant melanoma (M-6) cell line. Binding studies in M-6 membranes showed the coexistence of 2-[125I]iodomelatonin binding sites with picomolar and nanomolar affinities. Guanine nucleotides caused conversion of all high-affinity sites to a low-affinity state without a change in binding capacity. Melatonin induced a marked concentration-dependent reduction in forskolin-stimulated cAMP accumulation in intact M-6 cells, indicating that it binds to a functional receptor in this cell line. The in vitro proliferation of M-6 cells was significantly inhibited by melatonin and its analogues 6-chloromelatonin, and 2-iodomelatonin, at concentrations ranging from 10(-9) to 10(-4) M, as demonstrated by cell counts and measurements of DNA content. These findings indicate that M-6 cells express functional receptors for melatonin which may be involved in mediating the antiproliferative effects of this hormone.

Physiological neuroprotection by melatonin in a 6-hydroxydopamine model of Parkinson's disease.

There is considerable evidence that pharmacological doses of the pineal hormone, melatonin, are neuroprotective in diverse models of neurodegeneration including Parkinsons disease. However, there is limited information about the effects of physiological doses of this hormone in similar models. In this study, rats were chronically treated with melatonin via drinking water following partial 6-hydroxydopamine lesioning in the striatum. The two doses of melatonin (0.4 microg/ml and 4.0 microg/ml) were within the reported physiological concentrations present in the serum and cerebrospinal fluid respectively. At 2 weeks after surgery, the higher dose of melatonin significantly attenuated rotational behavior in hemi-parkinsonian rats compared to similarly lesioned animals receiving either vehicle (P < 0.001) or the lower dose of melatonin (P < 0.01). Animals were perfused or sacrificed 10 weeks after commencing melatonin treatment for immunohistochemical or mRNA studies. Animals treated with 4.0 microg/ml melatonin exhibited normal tyrosine hydroxylase (TH) immunoreactivity in the lesioned striatum, whereas little or no TH immunofluorescence was visible in similarly lesioned animals receiving vehicle. In contrast, semiquantitative RT-PCR analysis revealed no group differences in TH mRNA, suggesting spontaneous recovery of this transcript as observed previously in partially lesioned animals. There were no significant differences in striatal GDNF mRNA levels between sham and lesioned animals. However, there was a significant (P < 0.01) increase in GDNF mRNA expression in the intact contralateral striata of lesioned animals treated with vehicle. Interestingly, melatonin treatment attenuated this novel compensatory contralateral increase in striatal GDNF expression, presumably due to its neuroprotective effect. These findings support a physiological role for melatonin in protecting against parkinsonian neurodegeneration in the nigrostriatal system.

Epigenetic targets for melatonin: induction of histone H3 hyperacetylation and gene expression in C17.2 neural stem cells

Abstract:  We have reported the induction of glial cell line‐derived neurotrophic factor, a potent survival factor for dopaminergic neurons, in the C17.2 neural stem cell line following in vitro treatment with melatonin. Furthermore, we have detected the melatonin MT1 receptor in these cells. Given these findings and recent evidence that melatonin may play a role in cellular differentiation, we examined whether this indoleamine induces morphological and transcriptional changes suggestive of a neuronal phenotype in C17.2 cells. Moreover, in order to extend preliminary evidence of a potential role for melatonin in epigenetic modulation, its effects on the mRNA expression of several histone deacetylase (HDAC) isoforms and on histone acetylation were examined. Physiological concentrations of melatonin (nanomolar range) increased neurite‐like extensions and induced mRNA expression of the neural stem cell marker, nestin, the early neuronal marker β‐III‐tubulin and the orphan nuclear receptor nurr1 in C17.2 cells. The indoleamine also significantly increased mRNA expression for various HDAC isoforms, including HDAC3, HDAC5, and HDAC7. Importantly, treatment with melatonin for 24 hr caused a significant increase in histone H3 acetylation, which is associated with chromatin remodeling and gene transcription. Since the melatonin MT2 receptor was not detected in C17.2 cells, it is likely that the MT1 receptor is involved in mediating these physiological effects of melatonin. These findings suggest novel roles for melatonin in stem cell differentiation and epigenetic modulation of gene transcription.

Melatonin receptor mRNA expression in human granulosa cells.

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1alpha) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Pharmacological characterization of melatonin binding sites in Syrian hamster hypothalamus

The radioligand [125I]iodomelatonin was used to study melatonin binding sites in Syrian hamster hypothalamus and hippocampus. Scatchard analysis revealed a single binding site with nanomolar affinity in hypothalamus (Kd = 1.8 +/- 0.3 nM, Bmax = 75 +/- 7 fmol/mg protein; n = 4) and hippocampus (Kd = 2.2 +/- 0.2 nM, Bmax = 49 +/- 5 fmol/mg protein; n = 4). The Kd value calculated from the association and dissociation rate constants in hypothalamus was (k-1/k1) = 2.4 nM. Regional studies revealed that the highest binding of [125I]iodomelatonin occurs in the hypothalamus. Only indoles structurally related to melatonin exhibited significant affinity at this site. Prazosin was found to be a potent inhibitor of [125I]iodomelatonin binding in all brain regions studied. The pharmacological profile of this binding site indicated it to be unique, since serotonergic, dopaminergic and adrenergic drugs (other than prazosin) did not have appreciable affinity for it. Although saturation studies revealed only one binding site, the low Hill coefficients obtained for several inhibitors suggest that [125I]iodomelatonin labels multiple sites (or affinity states) in the hypothalamus.

Physiological regulation of melatonin receptors in rat suprachiasmatic nuclei: diurnal rhythmicity and effects of stress

A marked diurnal variation in high-affinity binding of 2-[125I]iodomelatonin ([125I]MEL) in rat brain sections containing the suprachiasmatic nuclei (SCN) was observed. Binding was highest late in the light phase and lowest during darkness, in inverse correlation to the serum melatonin rhythm. Interestingly, only high-affinity sites were present during most of the light phase while both high- and low-affinity sites were detected just before and during darkness. Guanosine triphosphate (GTP) in combination with sodium converted all high affinity sites to a low affinity state suggesting that the two sites observed during darkness represent the two states of the melatonin receptor. Acute swim-stress caused a significant elevation of serum melatonin, together with a decrease in the density of [125I]MEL binding in the SCN. The inverse relationship between circulating melatonin levels and binding, under two different physiological conditions, indicates that this hormone is involved in regulating its own receptors in the SCN.

Melatonin enhancement of [3H]-γ-aminobutyric acid and [3H]muscimol binding in rat brain

Abstract The pineal hormone, melatonin, enhanced the sodium-independent binding of [3H]-γ-amino-butyric acid ([3H]GABA) and [3H]muscimol in the rat cerebral cortex in vitro. This effect was augmented by preincubation of synaptic membranes with melatonin but was abolished by preincubation with Triton X-100. Saturation binding studies using [3H]GABA (2.5 to 1000 nM) indicated that the melatonin-induced enhancement of binding is due to an increase in low-affinity GABAA binding sites. These findings suggest that the central effects of melatonin involve modulation of GABAergic function.

Induction of GDNF mRNA expression by melatonin in rat C6 glioma cells.

In order to determine the physiological effect of melatonin on glial cell line-derived neurotrophic factor (GDNF), which is reportedly up-regulated by high doses of this hormone, concentration-dependent studies were carried out in cultured cells. RT-PCR studies indicated that, in addition to GDNF, rat C6 glioma cells express both of the G protein-coupled melatonin receptor subtypes, MT1 and MT2. When C6 cells were treated with physiological (0.05–1 nM) or higher (10 and 100 nM) concentrations of melatonin for 24 h, a significant induction of relative GDNF mRNA levels (n  = 4) was detected by semi-quantitative RT-PCR. These findings suggest that induction of GDNF is involved in physiological neuroprotection by melatonin. Given the potency of GDNF in maintaining nigrostriatal dopaminergic integrity, understanding the mechanisms of its induction by melatonin could provide novel therapies for Parkinsons disease.