Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers

Background In 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. Results RT-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. Conclusions The 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 Parkinson's disease.

in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.

Background
Neural stem cells are multipotent cells which are capable of self-replication and differentiation into neurons, astrocytes or oligodendrocytes in the central nervous system [1]. Because of their intrinsic plasticity and multipotency, there are great expectations that NSC transplantation will ultimately provide immense benefits in the treatment of neurodegeneration. However, it is essential to fully understand the cellular and molecular mechanisms involved in the differentiation and function of NSCs, in order to fully harness their therapeutic potential. Because of the very limited availability of NSCs in the central nervous system (CNS), neural stem cell lines are very useful for the study and characterization of NSC biology. For example, transplantation studies with the C17.2 neural stem cell line [2] have revealed that these cells express diverse neurotransmitter phenotypes, depending on the environment prevailing in the CNS area of engraftment [3,4]. Recently, transplanted C17.2 NSCs, genetically modified to express glial cell line-derived neurotrophic factor (GDNF), were found to engraft in the 6-hydroxydopamine-lesioned mouse striatum and to express therapeutic levels of this neurotrophin, with consequent protection of dopaminergic neurons in this model of Parkinson's disease [5]. Although this and other similar approaches are promising, limitations including the stability and regulation of transduced genes await resolution. Therefore, it was of interest to determine whether C17.2 cells have the intrinsic ability to express neurotrophins or neurotrophic factors, which would make them amenable to modulation by appropriate agents in vitro or in vivo. In addition, we examined whether these NSCs express receptors for the pineal hormone melatonin, which can induce GDNF mRNA and protein expression [6,7] and which has been implicated in the development of vertebrates including humans [8][9][10]. Initially, different concentrations and types of sera were used for cell culture in order to select optimal conditions for gene expression studies. We now report that C17.2 NSCs exhibit heterogeneous phenotypes and express neurotrophic factors and melatonin MT 1 receptors.

Effects of culture conditions on neurotrophic factor and cell-specific marker mRNA expression in C17.2 NSCs
Following two days in culture, C17.2 cells remain in an undifferentiated state, as indicated by their flat and rounded appearance (Fig. 1A) and high expression of the stem cell/progenitor cell marker, nestin (Fig. 1C,1E,1G). These cells also expressed the early neuronal marker, β-tubulin III, but there was little or no expression of the mRNA for the glial marker, glial fibrillary acidic protein (GFAP). After seven days in culture, differentiating C17.2 cells exhibit an elongated shape with an extension of neurite-like processes, as shown in Fig. 1B. However, as observed in undifferentiated cells after two days, there was still strong expression of nestin and β-tubulin III, with little or no detectable GFAP mRNA (Fig. 1D,1F,1H). An examination of neurotrophin mRNA expression in undifferentiated C17.2 cells, revealed a robust expression of GDNF, BDNF and NGF, regardless of the type or concentration of serum used for culturing ( Fig. 1C,1E,1G). A similar strong expression of BDNF and NGF was observed in differentiating cells after seven days, but GDNF mRNA was relatively lower in cells maintained in 1% fetal bovine serum (FBS) or 10% FBS + 5% horse serum (Fig. 1F,1H). The mRNA levels of the control gene, GAPDH, did not change under the conditions examined (Fig. 1I).

Detection of MT 1 receptor mRNA and protein in C17.2 NSCs
Melatonin MT 1 receptor mRNA was detected by RT-PCR in NSCs maintained for two days, especially in cells cultured in 1% FBS, as shown in Fig. 2A. GAPDH mRNA expression did not change under the conditions examined (Fig. 2B). C17.2 NSCs maintained for indicated periods in 1% FBS or 10% FBS + 5% horse serum, expressed the MT 1 receptor protein, as revealed by western analysis (Fig. 2C,2D). Interestingly, when cells were cultured for 2-3 days, the MT 1 protein detected had a molecular weight of about 30 kDa, which is less than the predicted size of the mature receptor. However, when cells were cultured for 10-12 days, a mature MT 1 receptor of about 40-45 kDa was present, as shown in Fig. 2D. The MT 2 receptor transcript was not detected under any of the conditions used in this study.

Induction of GDNF mRNA expression by melatonin in C17.2 NSCs
In order to assess the potential functionality of the MT 1 receptor detected in C17.2 NSCs, the effect of low physiological concentrations of melatonin on GDNF mRNA expression was examined. Cells were grown as described in Methods and treated with melatonin or vehicle (0.001% DMSO) for 24 hours. Following RT-PCR analysis, GDNF mRNA levels were converted to optical density (OD) values and normalized to GAPDH OD levels, as reported previously [6]. After conversion of GDNF/ GAPDH OD ratios to percentage values, one-way ANOVA indicated a significant treatment effect (F 3,7 = 7.03, p < 0.04). A Neuman-Keuls test indicated a significant increase in relative GDNF mRNA expression in cells treated with 0.05, 0.1 and 1 nM melatonin as shown in Figure 5.

Discussion
The expression of nestin in undifferentiated C17.2 cells is consistent with the presence of this intermediate filament protein in stem and progenitor cells in the mammalian CNS [11]. However, as noted above, nestin mRNA was also readily detected in cells exhibiting morphological changes characteristic of differentiation, after one week in culture. Similarly, mRNA for the early neuronal marker, βtubulin III, was found under all conditions examined, whereas GFAP mRNA was detected only in some cultures. These observations suggest that the C17.2 cells examined in this study are an heterogeneous population of stem and progenitor cells in keeping with evidence that NSCs Morphology and expression of neurotrophins and pheno-typic markers in C17.2 NSCs
The expression of diverse neurotrophins by NSCs is consistent with the role of these factors in the differentiation and development of the CNS. Presumably, the robust mRNA expression observed, particularly in cells maintained in 10% FBS + 5% HS, is driven by the serumenriched milieu of potential inducers including neurotransmitters, hormones and growth factors, such as basic fibroblast growth factor and epidermal growth factor, which can stimulate C17.2 cell growth in vitro [14]. In contrast to BDNF and NGF, which exhibited strong mRNA expression under all conditions examined, GDNF expression was weaker or not detectable in differentiating cells after seven days in culture. The suppression of GDNF expression might have been due to the prolonged exposure of NSCs to regulatory factors in the serum, as its decline appears to be inversely correlated with the concentration or enrichment of serum used for cell culture. Thus, moderate, weak or no expression of GDNF was observed in cells cultured for 1 week in 1% CS, 1% FBS or 10% FBS + 5% HS, respectively (see Fig. 1D,1F,1H). Various biological agents or pathways have been implicated in the regulation of GDNF expression. For example, fibroblast growth factor-2 and proinflammatory cytokines such as interleukin(IL)-1β, IL-6 and tumor necrosis factor-α stimulate GDNF synthesis and secretion [15]. Activation of protein kinase C by phorbol esters increases GDNF expression [15,16], whereas the adenylate cyclase activator, forskolin, inhibits GDNF production in cultured cells, suggesting an inhibitory role for the cyclic AMP-protein kinase A pathway [15]. The cAMP pathway and its transcriptional factor cAMP response element binding protein (CREB) have been shown to induce differentiation in neuronal progenitor cells [17,18]. Therefore, it is possible that activation of this pathway was involved in both the initiation of differentiation and the inhibition of GDNF expression observed in C17.2 cells after seven days.
While this work was in progress, it was reported that C17.2 neural stem cells constitutively secrete BDNF, GDNF and NGF, but do not label for GFAP or neuronal markers like β-tubulin III [19]. Our findings are in agreement with these observations with regard to neurotrophin expression. However, in contrast to their findings, β-tubulin III mRNA and immunoreactivity were readily detected in our study. In addition, although GFAP mRNA was weakly expressed or not detectable in some cultures, immunoreactivity for this glial cell marker is present in C17.2 cells, as shown in Figure 4. These differences may be due to our examination of β-tubulin III and GFAP expression in cells maintained for 2-12 days in culture, whereas their C17.2 cells were examined after 2-3 weeks [19]. Other factors, such as our use of low serum concentrations, as compared with the enriched culture medium used by Lu et al. [19], may also be involved. The detection of melatonin MT 1 receptor mRNA in C17.2 cells after 2 days but not after 7 days, presumably involves downregulation of this receptor. There is considerable evidence that many G protein-coupled receptors are downregulated by their agonists [20]. More importantly, melatonin, which is present in serum, has been found to suppress MT 1 transcription in vitro [21]. Interestingly, our immunocytochemical studies revealed MT 1 immunoreactivity within C17.2 cell bodies and extensions, as shown in Figure Melatonin  [20], it is also possible that the immunoreactivity detected within these neural stem/progenitor cells is due to the presence of newly synthesized MT 1 receptors. In accordance with this view, the MT 1 protein detected in short-term (2-day) cultures is about 30 kDa, which is less than the approximately 37-45 kDa molecular weight observed in various mammalian tissues [22][23][24]. Moreover, when cells were cultured for 10-12 days, a MT 1 receptor of about 40-45 kDa was detected, as shown in Fig. 2D. The mammalian MT 1 contains two glycosylation sites in its N-terminal [24] and it may exist in more than one glycosylated form, as has been reported for other G protein-coupled receptors [25,26]. Thus, the above cytochemical observations suggest that newly synthesized immature MT 1 receptors,which have yet to undergo posttranslational modification and translocation to the plasma membrane, were detected in cells cultured for 2 days in 1% FBS, whereas a mature glycosylated receptor was present in cells grown for longer periods. Although the MT 2 receptor transcript was not detected under any of the conditions used in this study, additional studies are required before the possibility of its expression in these cells can be ruled out. It is possible that MT 2 mRNA may undergo rapid turnover/degradation, while a functional protein may still be present. This is the first evidence that melatonin receptors are expressed in neural stem or progenitor cells and raises the obvious question of whether this hormone plays a role in neuronal development. Although studies in this field are limited, there is increasing evidence that melatonin is involved in the early development of vertebrates. For example, melatonin is produced in chick embryos as early as the 7 th day of embryonic development [27], and a physiological concentration of this hormone has been shown to significantly enhance mouse embryogenesis in vitro [8].
Colocalization of the MT 1 receptor with phenotypic markers in C17.2 NSCs Similarly, when sheep blastocysts were treated with melatonin for 24 hr in vitro, there was a significant increase in the percentage of embryonic survival [28]. Other studies have shown that functional G i protein-coupled melatonin receptors, which mediate inhibition of the adenylatecyclase-cAMP pathway, are present in the embryonic (day 8) neural retina [29]. Melatonin receptor transcripts for all the known G i protein-coupled receptor subtypes have been found in 24 hr-old embryos from Japanese quail [30]. Various studies have detected melatonin receptors in human fetal brain [31,32] and peripheral tissues [33]. Moreover, recent autoradiographic and in situ hybridization studies indicate that the melatonin MT 1 receptor is expressed in diverse areas of the human fetal brain [9]. Thus, the presence of MT 1 receptors in NSCs is in keeping with the foregoing, and supports the view that melatonin is involved in neurodevelopment. Colocalization evidence that the MT 1 receptor is present in both neural and glial progenitor cells is consistent with a neurodevelopmental role for melatonin, and suggests that in addition to the presence of the MT 1 in mammalian neurons [34], it may also be expressed in astrocytes, as observed in similar cells from rat [6] and chick brain [35]. The detection of nestin in some cells expressing the MT 1 receptor is consistent with its presence not only in neural progenitor cells but also in GFAP positive glial progenitors [36]. Preliminary evidence that melatonin induces GDNF mRNA expression in C17.2 NSCs, as we have observed previously in C6 glioma cells [6], supports the foregoing as this neurotrophic factor plays a critical role in both central and peripheral neurodevelopment [37,38]. GDNF also exerts neuroprotective effects in the CNS, including a potent role in the survival of dopaminergic neurons in the midbrain [39,40]. Therefore, modulation of GDNF expression may be one of the mechanisms underlying physiological neuroprotection by melatonin in the CNS [6].

Conclusions
In summary, the NSCs utilized in this study exhibited an intrinsic ability to express neurotrophins under various cell culture conditions. This ability was not affected by their morphological state, except in the case of GDNF mRNA expression which was lower in cells undergoing differentiation in FBS-supplemented media. Novel evidence that neural stem/progenitor cells express MT 1 receptors adds to the increasing evidence that NSCs can respond to diverse modulators [41], and suggests an early role for melatonin in CNS development. Moreover, since melatonin induces GDNF expression in NSCs, its potential in vivo modulation of this and/or other neurotrophic factors, via its G protein-coupled receptors in the brain or on transplanted NSCs, could have important implications for optimizing therapeutic strategies in neurodegenerative disorders such as Parkinson's disease.

Cell culture
The C17.2 cell line was derived by retrovirus-mediated oncogene (v-myc) transduction of cells from the external germinal layer of neonatal mouse cerebellum [2]. C17.2 cells were grown on 10 cm Corning culture dishes (Fisher Scientific Ltd., Nepean, ON, Canada) in DMEM supplemented with 2 mM glutamine and calf serum, fetal bovine serum or horse serum (Invitrogen Canada Inc., Burlington, ON) in the concentrations indicated. Cells were maintained in a humidified 5% CO 2 -95% air incubator at 37°C and routinely split at approximately 90% confluency [3].

RT-PCR
Total RNA was isolated from C17.2 cells with TRIzol as described by the supplier (Invitrogen Canada Inc., Burlington, ON). After DNase treatment, cDNA was synthesized from 1-2 µg of total RNA using the Omniscript reverse transcriptase kit (Qiagen Inc., Mississauga, ON) and oligo dT primers. PCR was carried out using 1.5 µl (or  (Table  1). Following a hot start at 95°C for 15 min, samples were amplified for 36 cycles (or 38 cycles for MT 1 and MT 2 ) at 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by a final incubation at 72°C for 10 min.

Treatment of C17.2 NSCs with melatonin
For semi-quantitative examination of the effects of melatonin on GDNF mRNA expression, cells were grown in 10% FBS + 5% horse serum (HS) for 1 week. After subculture, cells were kept in 10% FBS + 5% HS for 2 days followed by another subculture to 1% FBS for 2 or 3 days. The cells were then treated with vehicle (0.001% DMSO) or melatonin (0.05, 0.1, and 1 nM) for 24 hours. Following RNA extraction, RT-PCR was performed as described above, except that an annealing T m of 55°C was used and samples were amplified for 30 cycles. Glyceraldehyde 3phosphate dehydrogenase (GAPDH), was amplified with intron-spanning primers [42], in order to control for DNA contamination.

Immunocytochemistry
After fixation for 15 minutes in 4% paraformaldehyde on poly L-ornithine-coated glass cover slips, cells were incubated overnight at 4°C with anti-melatonin MT 1 1 and cell markers, a rhodamine (TRITC)-conjugated donkey anti-mouse IgG (1: 100; Jackson Immu-noResearch Labs. Inc.,West Grove, PA) was used to detect nestin, GFAP and β-tubulin III. Digital images were recorded on a Zeiss confocal microscope.