mGlu receptors are neurotransmitter receptors in the nervous system because they respond to synaptic glutamate and are involved in the regulation of synaptic plasticity (19
). However, the presence of this receptor in mESCs and its down-regulation during differentiation and up-regulation during neurogenesis suggests an additional role for mGlu receptors (27
). For example, the endogenous activation of this receptor is necessary for maintenance of self-renewal properties in mESCs (17
). Initially the presence of mGlu5 has been investigated by immunostaining and RT-PCR analysis to prove its presence in mESCs. These observations are in agreement with previous reports that have confirmed the expression of mGlu5 on mESCs.
Previous studies have suggested that expression of mGlu5 decreases during EB formation, followed by an increase during neurolation (17
). The results of mGlu5 in the current study are consistent with these reports. In this study we have also shown that treatment with the IC50 of MDMA (16
) during neural precursor formation and differentiation significantly decreased expression of the mGlu5 receptor. However treatment with MDMA during neural differentiation (post-plating) did not affect the expression of mGlu5. These results have suggested that MDMA may affect mGlu5 expression during neural precursor cell formation but not during differentiation. It is important to note that in our previous study we have also shown that the IC50 of MDMA significantly decreased neural markers, MAP2 and nestin expression only during neural precursor cell formation (group 1) and not during differentiation (group 2) (16
). Therefore, this suggests that MDMA may induce inhibition of neural precursor formation via down-regulation of the mGlu5 receptor, but this requires additional confirmation.
To further investigate the influence of this receptor and MDMA on stemness characteristics we applied glutamate, it’s natural agonist, during stem cell culture in the presence of LIF and absence of a feeder layer. The results revealed a higher expression of SSEA1. In a preliminary study we showed that glutamate depletion for 24 hours by glutamate transaminase reduced Oct4-GFP expression in mESCs. We showed that the IC50 dose of MDMA decreased the numbers of colonies despite the increased expression of SSEA1. These observations were further confirmed by flow cytometry. No substantial changes in Oct4 expression were noted, possibly because reduction of Oct4 requires more than four days (28
). Therefore, these results may imply a binary role for MDMA: reducing ESCs proliferation while maintaining or supporting self-renewal characteristics. In concordance with our results, such a role has been described for DA neurons. Previous studies have shown that MDMA, despite being a toxic drug, enhances the differentiation and survival of DA neurons both in vivo
and in vitro
). In other studies, such results have been obtained for glutamate in MSCs and neural progenitor cells in the brain (29
In order to confirm whether MDMA’s effects on stemness characteristic were mediated by the mGlu5 receptor, MPEP was applied prior to MDMA. MPEP partially reduced SSEA1 expression, which thereby suggested that this effect of MDMA was possibly mediated by the mGlu5 receptor in addition to other possible pathways. It has been reported that methamphetamines and ecstasy could motivate Janus kinas/signal transducers and activators of transcription (JAK/STAT) pathways; this effect is independent of the mGlu5 receptor and could not be reversed by MPEP (31
Previous research has reported that MPEP, itself, inhibited stemness characteristics (17
). In our experiment the expression of stemness markers in the MPEP group was similar to the culture of mESCs in the absence of a feeder layer and presence of LIF. The difference observed between the two studies could be attributed to different types of stem cells as the current study’s cells were feeder-dependent.
The reduced cell proliferation, despite the maintenance of stemness characteristics may be related to the toxic effect of MDMA. This effect might be induced by the activation of the caspase pathway, as observed by increased caspase activity in the MDMA group. It has been reported that MDMA can modulate an apoptotic pathway and induce oxidative stress both in vivo
and in vitro
). A study of the literature suggests that the activation of the mGlu5 receptor via protein kinase c (PKC) inhibits glycogen synthase kinase-3β (GSK3-β) (36
), thus mimicking the actions of the Wnt signaling pathway (37
) which regulates SSEA1 expression (38
) by inhibiting β-catenin phosphorylation. It may be expected that the addition of glutamate and MDMA increases the level of β-catenin and decreases its phosphorylated form, thereby maintaining self-renewal. However, this proposed mechanism needs further investigation. This is the first report which shows that MDMA can increase stemness characteristic in mESCs, elucidating the pathway that begins with MDMA-induced self-renewal and may allow us to better understand how MDMA reduces normal neuron loss that occurs during development.