Immunofluorescent staining with the primary antibody, mouse monoclonal anti-human A antibody (clone 6F/3D; Dako Deutschland GmbH, Hamburg, Germany), was performed on these sections. == Introduction == Alzheimer disease (AD)2is pathologically characterized by the extracellular deposits of amyloid peptide (A). A injures neurons in the neocortex and limbic system directly (1) and indirectly by triggering microglial release of various neurotoxic inflammatory mediators, including cytokines (tumor necrosis factor- and interleukin-1 (IL-1)) and reactive oxygen species (2). A is generated after Sorafenib serial digestion of Alzheimer amyloid precursor protein (APP) by the membrane-anchored -site APP-cleaving enzyme (BACE1, -secretase) and -secretase (3). It has been observed that knock-out of BACE1 or administration of the BACE1 inhibitor dramatically decreases A levels in the brain and attenuates behavioral and electrophysiological deficits in APP-transgenic mice (46). Thus, extensive investigations have focused on the direct inhibition of BACE1 to reduce A load in the AD brain; however , these studies have unfortunately not yet led to any efficacious therapy for AD patients due to the various physiological roles of BACE1 (7). Using alternative methods to inhibit BACE1 might be a preferable investigative approach. Inflammatory activation might lead to up-regulation of neuronal BACE1 expression in the AD brain, as NF-B signaling enhances (8), and PPAR activation suppresses (9), the activity ofbace1gene promoter. Accumulating evidence has shown that posttranslational modification of BACE1 is extremely important for the activity, intracellular trafficking, and lysosomal degradation of BACE1. For example , phosphorylation of BACE1 at Thr-252 by p25/Cdk5 increases the secretase activity (10), and phosphorylation at Ser-498 facilitates retrograde transport of BACE1 from endosomes to the trans-Golgi network (11). Ubiquitination at Lys-501 targets BACE1 to late endosomes/lysosomes for degradation (12). Finally, bisectingN-acetylglucosamine modification blocks delivery of BACE1 to lysosomes (13). p38 mitogen-activated protein kinases (p38 MAPKs) are a class of mitogen-activated protein kinases that are responsive to stress stimuli, such as inflammatory cytokines and reactive oxygen species. Phosphorylation of p38 MAPK has been observed in the postmortem brain in the early stages of AD (Braak CACH6 stages IVV) (14, 15). A and glutamate have each been shown to activate p38 MAPK in cultured neurons by increasing reactive oxygen species (16, 17), and the A-triggered microglial release of inflammatory mediators, especially IL-1, is hypothesized to activate neuronal p38 MAPK (1820). p38 MAPK mediates A-initiated microglial inflammatory activation (21, 22), phosphorylates Tau protein in neurons (20, 23, 24), and mediates A-induced synaptic impairment in the cultured hippocampal slice (25). However , no experiments have yet elucidated whether p38 MAPK phosphorylates BACE1 and regulates A generation. Previous studies demonstrating the effects of p38 MAPK in Sorafenib hippocampal slices or APP-transgenic mice used p38 MAPK inhibitors, which cannot distinguish neuronal p38 MAPK effects and microglial p38 MAPK effects Sorafenib (22, 25). In attempting to dissect the role of p38 MAPK in AD, it is essential to distinguish between p38 and p38 MAPK enzymes, as they have different functions (26). To investigate the pathogenic function of neuronal p38 MAPK, we used Cre-Lox or knockdown techniques to ablate p38 MAPK specifically in neurons and examined the effect on BACE1 degradation in this study. == Experimental Procedures == == == == == == Animal Models and Cross-breeding == APP/PS1 double transgenic mice over-expressing human mutated APP (KM670/671NL) and PS1 (L166P) under Thy-1 promoters (27) were kindly provided by M. Jucker, Hertie Institute for Clinical Brain Research, Tbingen, Germany; p38fl/flmice with loxP site-flankedmapk14gene were imported from BioResource Center, RIKEN Tsukuba Institute, Japan (28); and Nex-Cre mice expressing Cre recombinase from the endogenous locus of thenexgene that encodes a neuronal basic helix-loop-helix (bHLH) protein were kindly provided by K. Nave, Max-Planck-Institute for Medicine, Gttingen, Germany (29). All three mouse strains were on a C57BL6 genetic background. APP-transgenic mouse models with deletion of p38 MAPK specifically in neurons of the neocortex and hippocampus.