Sera directed the research and wrote the manuscript. Tau toxicity. These three different sites, Ser238, Thr245, and Ser262 were tested either by obstructing their phosphorylation, by Ser/Thr to Ala substitution, or pseudophosphorylation, by changing Ser/Thr to Glu. We validate the hypothesis that phosphorylation at Ser262 is necessary for Tau-dependent Schisandrin C learning deficits and a facilitatory gatekeeper to Ser238 profession, which is linked to Tau toxicity. Importantly we reveal that phosphorylation Schisandrin C at Thr245 functions as a suppressive gatekeeper, avoiding phosphorylation of many sites including Ser262 and consequently of Ser238. Therefore, we elucidate novel relationships among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity. and in cells, but whether they also target Tau and under what conditions remains mainly elusive (Hosoi et al., 1995; Hong et al., 1997; Zheng-Fischhofer Schisandrin C et al., 1998; Gong et al., 2005). Interestingly, studies indicate that Tau phosphorylation might be primed by profession of a particular site before Schisandrin C the event of additional phosphorylations (Hanger et al., 2009). Tau consists of multiple intrinsically disordered areas (IDRs), which interfere with structural stability of the protein (Uversky, 2015). Then, this gatekeeper phosphorylation effect suggests that it may enable or inhibit local tertiary constructions that expose or occlude additional, often distant, phosphorylation sites (Jeganathan et al., 2008; Sibille et al., 2012; Schwalbe et al., 2015). Hence, the effect of specific phosphorylations may be to regulate this structural plasticity of Tau, contribute to the subcellular localization of Tau isoforms (Sotiropoulos et al., 2017) and modulate their practical properties (Xia et al., 2015). Irrespective of whether it is mutated, or wild-type, pathological Tau presents improved phosphorylation at sites occupied physiologically, but also on sites occupied only when pathology is present and are referred to as disease-associated epitopes (Morris et al., 2015; Arendt et al., 2016). Even though mechanisms that result in hyper-phosphorylation are unclear at present, the result is definitely neuronal deposition of hyper-phosphorylated Tau (Martin et al., 2011). If indeed phosphorylations modulate the structure and practical properties of Tau Bmp7 isoforms, then this hyper-phosphorylation is likely to underlie significant changes in the Schisandrin C properties of the protein that underlie its pathobiology (Regan et al., 2017). In fact, extensive literature offers led to the widely held notion that aberrant Tau phosphorylation is definitely central to neuronal pathology (Stoothoff and Johnson, 2005) and offered evidence that soluble hyper-phosphorylated Tau contributes to neuronal dysfunction before its aggregation (Fath et al., 2002; Santacruz et al., 2005; Brandt et al., 2009; Decker et al., 2015). Antibodies that identify non-physiologically phosphorylated Tau at specific sites (phosphoepitopes) in patient neurons but not in age-matched healthy individuals have been developed and used as specific diagnostic markers of Tauopathies (Sergeant et al., 2005). However, the mechanistic understanding of the sequential phosphorylation events that happen on Tau and which sites are essential for maintenance and development of pathology are still unclear. Recognition of phosphorylation sites on Tau that either result in or are essential for pathogenesis are pivotal to our understanding of Tau-dependent neuronal malfunction and toxicity. Drosophila models of Tauopathies contribute significantly to the concept that build up of prefibrillar hyper-phosphorylated forms of Tau correlate with human being Tau-mediated toxicity in flies (Wittmann et al., 2001; Steinhilb et al., 2007a, b; Feuillette et al., 2010). Recently, we have recognized two novel phosphorylation sites on Tau, Ser238 and Thr245, as essential for its harmful effects on mushroom body (MB) integrity (Kosmidis et al., 2010; Papanikolopoulou et al., 2010) and premature lethality (Papanikolopoulou and Skoulakis, 2015). The MBs are neuronal assemblies that constitute major insect mind centers for learning and memory space (Davis, 2005). Significantly, obstructing Ser238 and Thr245 phosphorylation by substituting them with alanines (STA mutant), yielded animals with structurally normal but profoundly dysfunctional MBs, as flies accumulating the mutant protein exhibited impaired associative learning (Kosmidis et al., 2010). Moreover, our results strongly suggested that Ser238 profession is a critical mediator of Tau neurotoxicity in standard wheat-flour-sugar food supplemented with soy flour and CaCl2 and cultured at 25C and 50C70% moisture inside a 12 h light/dark cycle unless noted normally. The Elavc155-Gal4 and Ras2-Gal4 have been explained before (Gouzi et al., 2011). The Elav-Gal4 collection on the second chromosome was from Bloomington Drosophila Stock center (#8765). The dual Gal 4 driver strains Elavc155-Gal4;Ras2-Gal4 (henceforth Elav;Ras2) and Elavc155-Gal4;Elav/CyO (henceforth Elav;Elav), were constructed by standard crosses. The hTau0N4(0N4R) transgenic flies were a gift from M. Feany.