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    • On the other hand with the

    2018-11-12

    On the other hand, with the present study we verified that METH did not alter protein expression of cyclin inhibitors, p21 and p27, which suggests the involvement of other mechanisms in METH-induced cyclin E down-regulation. In fact, Heo et al. (2006) showed that blockade of MAPK pathway with PD-98059 (MEK1 inhibitor) decreased both cyclins D1 and E protein levels. These authors investigated the involvement of MAPK pathway activation in cell proliferation by stimulating mouse embryonic stem cells with epidermal growth factor (EGF), which induced an increase in the phosphorylate state of ERK1/2 and cyclin E proteins (Heo et al., 2006). Furthermore, homocysteine, a sulfur-containing intermediate of methionine metabolism, decreased phosphorylation of ERK1/2 and cyclin E protein expression in mice SVZ cells incubated with FGF-2 (Rabaneda et al., 2008). Based on our results, we may suggest that METH acts upstream to ERK1/2 by decreasing the phosphorylation levels of EGFR, which will interfere with the MAPK pathway through down-regulation of pERK1/2 followed by a decrease in cyclin E expression, that in turn will affect RPC1063 progression and resulting in the impairment of DG stem cell self-renewal. In fact, little is known regarding the effect of METH on EGFR, but it was previously demonstrated that neonatal EGF administration increased METH-induced locomotor responses (Mizuno et al., 2004). Still, it was shown that EGFR plays an active role in promoting cell proliferation and neuronal differentiation in the SGZ, as well as in rescuing neurogenesis in aged rats (Jin et al., 2003). Additionally, in a Parkinson\'s disease animal model, EGFR expression is down-regulated in proliferating cells in the SVZ (Höglinger et al., 2004). Furthermore, it was demonstrated that EGFR expression and signaling were reduced in the SVZ of aged mice, which resulted in a decrease of cell proliferation and neurogenesis (Enwere et al., 2004). Consistent with this finding, we suggest that METH decreases the expression of pEGFR, which down-regulates MAPK signaling as verified by a transient decrease of ERK1/2 phosphorylation, which could lead to down-regulation of cyclin E and, consequently, a decrease in DG stem cell proliferation and self-renewal. Although MAPK pathway can mediate cell proliferation, it can also be involved in memory performance. Indeed, depending on the frequency of METH exposure, an acute (Cao et al., 2013) or chronic (Ito et al., 2007) administration can improve or decline memory performance, accompanied with an increase or decrease in the phosphorylation levels of ERK1/2, respectively. Furthermore, ERK1/2 activation leads to the activation of the downstream transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) followed by increased expression of c-fos, which in the DG identifies neurons processing spatial information (Sweatt, 2001). Noteworthy, activation of ERK1/2 is necessary to generate long-term potentiation in DG immature neurons (Darcy et al., 2014). Interestingly, Kee et al. (2007) verified that immature neurons present increased c-fos expression, which strongly suggest that these cells are recruited to integrate spatial memory circuitries. Accordingly, in the present study we demonstrate that METH transiently decreased pERK1/2 protein levels in DG stem cells and increased immature neurons, which allow us to hypothesize that these neurons can be synaptically active and thereby participate in memory processes. Other important characteristic of neural stem cells is the ability to self-renew. Thus, we further explored for the first time the effect of METH on DG stem cell self-renewal. METH, at nontoxic concentrations (1nM or 10nM), was able to decrease the number of primary neurospheres, showing that this drug has a negative effect on stem cell proliferation. In fact, the findings obtained in cell cycle analysis can be correlated with the decreased number of primary neurospheres. Specifically, METH delayed G0/G1-to-S phase progression which decreased the proliferative capacity of DG stem cells and, consequently, could decrease the number of neurospheres. Also, at the highest nontoxic concentration (10nM), METH decreased the number of secondary neurospheres proving that it affects DG stem cell self-renewal. Some studies addressed the effect of METH on DG neurogenesis and focused mainly on cell proliferation. In detail, it has been described that METH (25mg/kg) transiently decreases the number of BrdU-positive cells in gerbil dentate gyrus (Teuchert-Noodt et al., 2000). Moreover, Mandyam et al. (2008) observed that this drug (0.05mg/kg/infusion) administered 1h/day and 6h/day decreases the number of proliferating cells, characterized by the decreased number of Ki-67-positive cells, a protein expressed during the active phases of the cell cycle. Also, METH induces long-term effects in SGZ stem/progenitor cell proliferation, as described by Schaefers et al. (2009) showing that a single METH administration (50mg/kg) to 14-day-old gerbils decreases the number of BrdU-positive cells, 45days post-administration. In addition, our group demonstrated that 100μM METH (48h of exposure) reduced proliferation in SVZ cultures (Bento et al., 2011).