Primary antibody incubation was performed overnight at 4?C. phosphorylation were observed in tau knockout mice. On the contrary, addition of tau proteins promoted ERK dephosphorylation release, representing a pathological mechanism of E-NMDARs in AD development.11 Another study suggested a toxic exposure to glutamate enhances tau BAY 61-3606 dihydrochloride mRNA expression in primary neuronal cultures.13 However, as glutamate incubation activates both the synaptic and extrasynaptic NMDA receptors, the role of E-NMDARs in this process was not distinguished. In the present study, we explored the effect of E-NMDAR activation on tau expression and its role in neurodegeneration. We found that selective extrasynaptic but not synaptic NMDA receptor activation induced tau overexpression and neuronal degeneration/death in cultured primary neurons and mouse brain hippocampus, which could be BAY 61-3606 dihydrochloride reversed by pretreatment of memantine, an antagonist of E-NMDARs. In tau knockout (Ko) mice or neurons, selective activation of E-NMDARs failed to induce cell death, with retained surviving signaling ERK activation. BAY 61-3606 dihydrochloride Increased mitogen-activated and extracellular signal-regulated kinase kinase (MEK) activity, decreased binding and activity of ERK phosphatase to ERK, and increased ERK phosphorylation was observed in tau Ko mice, whereas addition of tau proteins into tau Ko mice brain homogenates promoted the ERK dephosphorylation control neurons, gene was analyzed according to the Ct method (comparative Ct method), in which Ct is the threshold cycle value and normalized by control). (e) Primary cortical mouse neurons (12C14 DIV) were treated with synaptic or extrasynaptic NMDA receptors activation HSPB1 protocols for 24?h, immunofluorescence staining images with Tau-1 (red) and MAP-2 (dendrite marker, green) were acquired under a confocal microscope. Scale bar=50 control group, control group; #extrasynaptic NMDAR activation group, control group, control group. (c) Wt mouse primary cortical neurons at 5 DIV were transfected with EGFP by lentivirus. At 12 DIV, neurons were subjected to extrasynaptic NMDAR activation for 24?h. Morphological changes of EGFP-labeled neurons treated with BAY 61-3606 dihydrochloride DMSO (Ctrl) BAY 61-3606 dihydrochloride or the extrasynaptic NMDA receptor activating protocol for 24?h (E-NMDAR). Images were acquired by confocal microscopy. White arrows showed abnormal neurodegeneration. (d) Representative neuron images from tau Ko mouse cortical neurons treated with DMSO (Ctrl) or E-NMDARs activation protocol (E-NMDAR) for 24?h, neurons were directly fixed and visualized under the fluorescence microscope. Scale bar=50?findings, we injected NMDA into the mouse hippocampus directly to induce extensive activation of NMDA receptors, including extrasynaptic NMDA receptor. We first evaluated the expression of tau after NMDA injection, the result showed a significant increase of total (Tau-5), phosphorylated (pS262) and dephosphorylated (Tau-1) tau levels in mouse brains compared with the saline-injected control group (Figures 3a and b), which was consistent with the changes observed in cultured primary neurons treated with E-NMDAR activation protocol. We then used Nissl staining to detect neuron survival of the three groups. Results showed NMDA injection induced significant neuron loss in CA2 and CA3 regions of the hippocampus of wild-type mice; whereas in tau Ko mice, NMDA injection did not reduce neuronal survival (Figures 3c and d).These results reinforced the idea that E-NMDAR activation triggers tau expression, and increased tau could promote neuronal degeneration and death. Open in a separate window Figure 3 Tau deletion protects neurons from E-NMDARs-triggered neuronal death in mouse hippocampus. (a) Wild-type (Wt) C57 mice were injected with saline (Ctrl) or NMDA (60?mM, 2?saline-injected control mice, Wt NS group. ##NMDA-treated Wt mice (wild-type neurons, #tau Ko control neurons, ##tau Ko control neurons, Wt neurons, ##tau Ko control neurons, Wt mice, Wt mice, Wt mouse brain homogenates, #tau Ko.