Osphorylates the wellcharacterized KxGS motif on Tau Serine 262 (S262) residue (Figure 5A). When coexpressed in cell lines, both LKB1 (coexpressed with its coactivator STRAD) and CAMKK2 are potent activators of AMPK, although we observed that CAMKK2 was drastically far more potent in phosphorylating AMPK on T172 than LKB1 or CAMKK1 (Figure 5B). Moreover, direct activation of AMPK working with the AMP analog AICAR triggered a dose-dependent increase of Tau phosphorylation of S262 in cortical neurons (Figures 5C, 5D, and S4), a therapy that induces a dose-dependent reduction in spine density (Figures 1N and 1O). The microtubule-associated protein Tau is phosphorylated in numerous websites (Mandelkow and Mandelkow, 2012), and analysis of six well-characterized phosphorylation internet sites revealed that following 24 hr therapy with AICAR, phosphorylation of Tau on S262 is considerably improved in a dose-dependent manner but that other web-sites are either unchanged (for example, the other KxGS motif on S356, also as S396, S422) or decreased (S202/T205, S404) (Figures S4A and S4B).TBB This observation suggests that S262 is definitely an critical target of AMPK, and phosphorylation of this website could possibly underlie AMPKinduced spine loss.Orexin 2 Receptor Agonist Stopping Tau Phosphorylation on S262 Protects Hippocampal Neurons from the Synaptotoxic Effects of A42 Oligomers In Vitro along with the Dendritic Spine Loss Observed within the APPSWE,IND Mouse Model In Vivo Prior research in Drosophila recommended that overexpression of AMPK-related member PAR-1/MARK2 induced neurotoxicity by way of phosphorylation of Tau in the microtubulebinding domains on S262 and S356 and that phosphorylation of these internet sites played an initiator role inside the pathogenic phosphorylation course of action of Tau (Nishimura et al.PMID:32261617 , 2004). Offered the importance of phosphorylation of S262 as a “priming” internet site (Biernat et al., 1993) plus the recent implication of Tau within the synaptotoxic effects of A42 oligomers (Ittner et al.,Neuron. Author manuscript; accessible in PMC 2014 April 10.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMairet-Coello et al.Page2010; Roberson et al., 2007), we wanted to test if expression of a type of Tau that cannot be phosphorylated on S262 could exert a protective impact inside the context of A42 oligomerinduced synaptotoxicity in cultured hippocampal neurons. Expression of Tau S262A abolished the loss of spines induced by A42 oligomers (Figures 5EH), despite the fact that its expression in handle neurons did not have any impact on spine density. By contrast, expression of Tau WT or even a phospho-mimetic version of Tau on S262 (Tau S262E) resulted in spine loss in handle situation, and the WT type of Tau was unable to prevent the synaptotoxic effects of A42 oligomers. Ultimately, the nonphosphorylatable form of Tau on S356 (S356A) displayed similar protective effects as Tau S262A mutant, indicating that the phosphorylation of those two serine residues within the microtubule-binding domains plays a essential part in mediating the synaptotoxic effects of A42 oligomers. To investigate the relevance from the phosphorylation of Tau on S262 in vivo, we performed in utero electroporation of Tau S262A construct in E15.5 WT and J20 embryos and analyzed spine density of CA3 hippocampal pyramidal neurons inside the adult mice at 3 months (Figures 5I and 5J). Tau S262A slightly decreased spine density in WT animals in comparison with manage vector, suggesting that phosphorylation of Tau on S262 plays a role in spine improvement. Nevertheless, T.
