(C) Lysates were prepared from PS1+/? and PS1?/? mouse embryonic brains. apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway. to yield an N-terminal (PS1/NTF) fragment and a C-terminal (PS1/CTF) fragment that associate to form a functional heterodimer (Thinakaran experiments showed that overexpression of PS1 FAD mutants promotes apoptosis (Weihl formation of the complexes would re-activate Akt. To this aim, we used a calcium switch approach to disrupt and re-form cadherin/PI3K complexes (Pece using PS1 null mice. Figure 6A shows that, compared to WT embryos, PS1?/? embryos contain significantly lower amounts of the p85/E-cadherin complexes, whereas an even more dramatic reduction is observed in the levels of the N-cadherin/p85 complexes. Phosphorylation of both Akt and its substrate GSK-3 is also reduced in PS1?/? embryonic brains compared to WT littermates, indicating reduced activation of the PI3K/Akt pathway and increased GSK-3 activity in the absence of PS1 (Figure 6B). Open in a separate window Figure 6 PS1 knockout embryos show reduced cadherin/PI3K complexes, decreased phosphorylation of Akt and GSK-3 and increased GSK-3-dependent phosphorylation of tau. (A) Total embryo homogenates prepared from PS1+/+ or PS1?/? mouse embryo littermates were immunoprecipitated with anti-E-cadherin (IP: E-cad) or anti-N-cadherin (IP: N-cad) antibodies and analyzed as shown. (B) Lysates were prepared from PS1+/+ or PS1?/? embryonic brains and analyzed for phosphorylated Akt and GSK-3 as shown. (C) Lysates were prepared from PS1+/? and PS1?/? mouse embryonic brains. The heat-stable fraction of lysates was analyzed with phosphorylation-dependent (PHF1, CP13) and phosphorylation-independent (TG5) anti-tau antibodies. Duplicate samples each from a littermate embryo are shown. GSK-3 (also called tau kinase 1) phosphorylates tau at several serine and threonine residues found hyperphosphorylated in AD brains (Hanger pathway To further explore the role of PS1 in POLB GSK-3-dependent phosphorylation of tau, we transfected PS1+/+ and PS1?/? fibroblasts with the longest human tau isoform and then examined phosphorylation of tau residues Ser396/404 and Ser202 that are targets of GSK-3 and are overphosphorylated in AD brains (Sperber FAD models. Figure 8C (panels aCd) shows that phosphorylation of both Akt and GSK-3 is reduced in the brains of knock-in mice. In agreement with the reduced phosphorylation, and hence increased activation, of GSK-3, tau protein is overphosphorylated in the knock-in mice (panels eCf). Importantly, co-immunoprecipitation experiments showed that cadherin/PI3K association is reduced in Pitolisant the FAD mutant knock-in mice (Figure 8D), supporting the suggestion that this mutation may reduce Akt phosphorylation and signaling by interfering with the ability of PS1 to promote cadherin/PI3K association. Together, our data show that PS1 FAD mutants Pitolisant are impaired in their ability to stimulate the PI3K/Akt pathway and to suppress AD-related tau overphosphorylation and activation of apoptotic caspase-3. Discussion Our data reveal a novel PS1 function by which this protein stimulates PI3K/Akt signaling and promotes cell survival. This conclusion is supported by the following observations: (1) absence of PS1 results in low Pitolisant levels of phosphorylated Akt and increased apoptosis; (2) exogenous PS1 stimulates Akt phosphorylation and rescues PS1 null cells from apoptosis; (3) a constitutively active PI3K restores Akt activation and suppresses apoptosis induced by the absence of PS1; (4) pharmacological inhibition of either PI3K or Akt prevents the PS1-dependent Akt phosphorylation and caspase-3 inactivation, indicating that the PI3K/Akt pathway mediates the anti-apoptotic effects of PS1. CadherinCcadherin interactions initiate a cascade of signaling events that result in increased cadherin/PI3K association, activation of PI3K/Akt signaling and increased cell survival (Pece activation of the cadherin/PI3K/Akt signaling and tau phosphorylation is provided by PS1 knockout mice, which show decreased cadherin/PI3K association, reduced PI3K/Akt activity, indicated by the decreased phosphorylation of Akt and GSK-3, and increased tau phosphorylation at AD-related residues. In agreement with the decreased activity of the PI3K/Akt cell survival pathway, PS1 null mouse embryos die at birth showing increased neuronal death, probably by apoptosis, and serious deformities (Shen and cell death detection kit, fluorescein’ (ROCHE). Determination of Pitolisant early apoptotis by flow cytometry was performed using the annexin Pitolisant VCPE apoptosis detection kit following the manufacturer’s directions.