Furthermore our lab previously reported that

Furthermore, our lab previously reported that cadmium treatment increased AChE-S and decreased AChE-R variants gene expression, which mediates the cell death observed on basal forebrain cholinergic neurons (Del Pino et al., 2016b). AChE-R decreased expression, which presents a neuroprotective role in neurodegenerative process (Berson et al., 2008), with an excess of the synaptic AChE-S has been associated with neurodeterioration (Birikh et al., 2003), supporting our previous results. We also recently showed that cadmium effect on AChE variants was mediated, in part, through blockade of muscarinic M1 receptor, but other mechanism seemed to be involved (Del Pino et al., 2016a). In the present work, we show that knockdown of M2R and M4R had not effect on AChE variants gene expression, but single or simultaneous knockdown of M1R and M3R decreases and increases the gene expression of AChE-R and AChE-S, respectively, being more pronounced this effect after simultaneous silencing of M1R and M3R. Our results suggest that both receptors regulate AChE variants expression in basal forebrain, confirming our previous data on M1R. Cadmium treatment of single or simultaneous M1R and M3R silenced PF-03814735 receptor induced a higher alteration of AChE variants than that observed after cadmium treatment alone, being this effect more pronounced after cadmium treatment of simultaneous M1R and M3R silenced cells. These results suggest that both receptors mediate in part the cadmium effect on AChE variants, although other mechanism should be involved. Besides, NAC treatment reversed partially this effect, which indicates that cadmium mediates, in part, its effect through oxidative stress, although its chelator properties could contribute to the observed effect. Previously, it has been reported that oxidative stress could alter AChE variants expression (Bond et al., 2006; Härtl et al., 2011; Toiber and Soreq, 2005), which supports our results. The alteration of AChE variants has been involved with induction of basal forebrain cholinergic neuron loss and cognitive disorders (Cohen et al., 2008; Del Pino et al., 2016b; Farchi et al., 2007), so their alteration induced by cadmium could mediate the cognitive disorder through induction of cell death in basal forebrain cholinergic neurons. Muscarinic M1 and M3 receptors are necessary to maintain cell viability (Budd et al., 2004; De Sarno et al., 2003; Giordano et al., 2009; Medeiros et al., 2011). M3R activation has been reported to prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells (Giordano et al., 2009), which supports that its downregulation mediates the effect observed on cell viability. ROS generation induced by Cd treatment, altered M1R and M3R, leading to basal forebrain cholinergic neurons viability reduction, but other mechanism seems to be involved. In this regard, cadmium has been reported to be a calcium channel blocker (Saroj et al., 2017; Zhou et al., 2015), and a blockade of calcium channels could induce cell death (Choong et al., 2014; Zhou et al., 2015), which could also contribute to the effect observed on basal forebrain. In addition, cadmium has been reported to block estrogen receptors (Chouchene et al., 2016) that are necessary to maintain redox status and cell viability of basal forebrain, and its blockade or deficiency has been reported to induce ROS, cell death and cognitive disorders (Kwakowsky et al., 2016; Lichtenfels et al., 2017; Mladenović et al., 2014; Wassmann et al., 2001), so this mechanism may also be involved. In addition, our previous research showed that cadmium M1R blockade induced cell death on SN56 cells mediated, in part, through increase GSK-3β gene expression and Aβ, total and phosphorylated Tau levels formation (Del Pino et al., 2016b). However, other mechanisms seem to mediate these actions. In this regard, muscarinic M3 receptor stimulation has been related with a reduction in the formation of Aβ, thus M3R blockade could induce its formation. Furthermore, ROS have been related with GSK-3β gene expression alteration, Aβ production and Tau abnormal phosphorylation (Bond et al., 2006; Giraldo et al., 2014; Rojo et al., 2008; Tamagno et al., 2005; Wang et al., 2009), which could also contribute to explain the induction of these mechanisms.