EMBO J. expressing excess amounts of human Alc CTF (hAlc CTF) in neurons, we found that expression of hAlc CTF induced excess production of hAlc ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including A generation. cell studies also demonstrated that excess expression of Alc ICD released both APP and Alc from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alc by -secretase regulates APP trafficking and the production of A gene knock-out (X11L-KO) mice showed enhanced generation of endogenous A in the brain (20), and human APP transgenic mice lacking the X11L gene exhibited enhanced amyloid plaque formation in the brain (21). However, the role of Alc metabolites in APP metabolism and A generation remain unclear indicate p3-Alc species (indicates p3-Alc35, a major p3-Alc species). Intensity; arbitrary unit. = 3). Specificity of the anti-Alc no. 958 antibody is shown in Fig. 2for 10 min, and a clarified supernatant (S1 fraction) was recovered. The S1 fraction was further centrifuged at 13,800 for 20 min, and the supernatant (S2 fraction) and pellet (P2 fraction) were used for assays. The P2 fraction was suspended again in buffer A and overlaid on a discontinuous sucrose gradient prepared with 1.2 m, 1 m, and 0.85 m sucrose solution and centrifuged at 82,500 for 2 h with an SW41 rotor (Beckman Coulter). After centrifugation, the layer between 1.0 m and 1.2 m sucrose was collected and Refametinib resuspended in 6 mm Tris-HCl (pH 8.0) buffer containing 0.5% (v/v) Triton X-100 to prepare the synaptosome fraction. Antibodies, Immunoprecipitation, and Immunoblot Analysis A rabbit polyclonal anti-Alc carboxyl-terminal domain antibody, no. 958, was raised against a synthesized peptide, 948GEQGDPQNATRQQQL962, of human Alc1. IgG was affinity purified with antigen-coupled beads and used for analyses. This antibody specifically recognizes Alc of human and mouse almost equivalently but does not show cross-reactivity with Alc and Alc Refametinib of human (see Fig. 2indicates FLAG-tagged Alc, Alc, and Alc, and the indicates Alc CTF. indicate protein standards (kDa). The indicates a nonspecific product. and and and 0.005, Student’s t test. #, below detectable levels. indicate hAlc ICD fragments. on the of the panel indicate the molecular mass (kDa). indicate IgG heavy and light chains. were quantified. The indicates the intensity of cell body, and the indicates the intensity of neurite. The fluorescence intensity of cell body or neurite is shown as a ratio to Rabbit Polyclonal to Cytochrome P450 26C1 average intensity of cell body plus neurite. (control) and (DAPT-treated) are shown. The values are indicated with mean S.E. (= 9C14). *, 0.05, Student’s test. sign, whereas a Refametinib indicates the use of empty plasmid alone. indicate the protein molecular mass standards. RESULTS Generation of Human Alc Carboxyl-terminal Fragment Transgenic Mice A cDNA sequence encoding the carboxyl-terminal region of human Alc1 together with the signal peptide sequence was expressed under the regulation of the mouse PDGF- promoter (Fig. 1(10). MALDI-TOF/MS analysis of p3-Alc secreated from HEK293 cells showed that the p3-Alc35 (amino acid sequence indicated in and and and and = 3, Fig. 2= 0.0017); TBS insoluble A1C42, non-Tg = 0.18 0.00 pmol/g tissue, Tg54 = 0.22 0.01 pmol/g tissue (= 0.0105)) (Fig. 3indicate statistical significance as determined by Student’s test (= 12, **, 0.01). with and on the represent protein molecular mass standards. on the indicate the protein detected with antibodies. The bar graphs show the results of quantification of protein bands in the total lysate (graph) and APP CTFs (graph) in the lysate and synaptosome fractions are indicated. The pC99 was also quantified, and the ratio of pC99/total CTFs (graph) is shown. Values were normalized to the values of flotillin-1. Values obtained from non-Tg mice were set to 1 1.0 and are given as the mean S.E. indicate statistical significance as determined by Student’s test (= 5; *, 0.05). Cleavage of APP in neurons occurs during or after its axonal transport; in other words, in the late secretory pathway. Therefore, the brains of Tg54 and non-Tg mice were fractionated to isolate the synaptosome fraction, which includes membrane vesicles of the late secretory pathway. As shown in Fig. 3= 5, = 0.0458) when the value in non-Tg mice was set at 1.0). However, total APP, which includes mAPP and immature APP, was not changed significantly in the total lysate of either Tg or non-Tg mice (lysate = 1.06 0.19, = 5, = 0.4904). APP CTFs were also significantly more abundant in the synaptosome fraction of Tg54 mice compared with non-Tg mice (1.27 Refametinib 0.09 (= 7, = 0.0261) when the value in non-Tg mice was.