 8.0), 300 mM NaCl, 30 mM imidazole, 10% glycerol, and 2 mM 2-mercaptoethanol. The suspensions were sonicated on ice and centrifuged at 15,000 ϫ g for 30 min at 4°C. The supernatants were incubated with nickel-nitrilotriacetic acid-agarose resin (50% slurry, Qiagen Japan, Tokyo, Japan) for 2 h at room temperature with shaking. Reduced proteins were loaded directly onto a PD-10 column (GE Health-Care UK Ltd.) equilibrated with G2 buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 5% glycerol. Protein concentrations were determined using spectrophotometry at 280 nm (⑀ 0.1% ϭ 1.0).
Congo Red Birefringence-The procedure for Congo red birefringence was carried out, as described previously (11), with minor modifications. Aliquots (40 l) of A␤40 at 50 M with or without 10% GAPDH aggregates were incubated for 48 h (day 2) and added to a 360-l Congo red solution (25 g/ml in PBS). This mixture was incubated for 30 min at 25°C and then centrifuged at 20,400 ϫ g for 30 min at 4°C. The resultant pellets were resuspended in 50 l of sterilized Milli-Q water and then dried on a glass slide. Birefringence was observed with an Eclipse LV100POL microscope equipped with a polarizing stage (Nikon, Tokyo, Japan).
Circular Dichroism (CD)-The far-ultraviolet CD spectrum of A␤40 at 50 M treated with or without GAPDH aggregates at day 2 was measured at 37°C with a spectropolarimeter (model J-820, Jasco, Tokyo, Japan). The path length of the optical quartz cuvette was 1.0 mm at 200 -250 nm. Spectra were obtained as the average of eight successive scans with a bandwidth of 2.0 nm. These spectra are expressed as values subtracted from that of each vehicle. These data are expressed as molar residue ellipticity ().
Atomic Force Microscopy (AFM)-Aliquots (10 l) of A␤40 at 50 M with or without GAPDH aggregates were incubated for 48 h (day 2), sonicated, and spotted onto freshly cleaved mica, incubated for 1 min, rinsed three times with water, and then dried. All measurements were carried out in "tapping mode" under ambient conditions using AFM (Nanoscope, Veeco Instruments Inc., Santa Barbara, CA) with single-beam silicon cantilever probes. We examined three regions of the mica surface to ensure that we obtained an accurate sample of structures on the mica (39).
Mitochondrial Membrane Potential Assay-Mitochondrial membrane potential was measured using rhodamine 123 (40). PC12 cells (3 ϫ 10 5 /cm 2 ) were seeded on glass coverslips coated with 0.1 mg/ml poly-D-lysine and grown for 24 h at 37°C. In this study, PC12 cells were divided into four treatment groups as follows: 1) control, vehicle of DMEM/F-12, 1% fetal bovine serum; 2) aggregated A␤40 (50 M) incubated for 48 h at 37°C in vehicle; 3) aggregated A␤40 (50 M) plus 10% GAPDH aggregates (5 M) in vehicle; and 4) 10% GAPDH aggregates in vehicle. After treatment, the culture medium was replaced with DMEM/F-12 containing rhodamine 123 (5 g/ml), followed by incubation for 10 min at 37°C in a 5% CO 2 -humidified incubator. After two washes with DMEM/F-12 and one wash with PBS, fluorescence from rhodamine 123-loaded cells was captured using a confocal scanning microscope (model C1si-TE2000-E, Nikon; excitation, 488 nm; emission, 525 nm). Raw images (500 ϫ 500 m) were first grayed and then transformed into binary images using Scion imaging software (version 4.0.3., Scion Corp.). The number of binary pixels in the square images was measured automatically. For semi-quantification of fluorescence, five microscopic fields were selected at random, and the number of cells among at least 500 total cells was quantified.
Animals-All animal experimental procedures were approved by the Animal Ethical Committee of Osaka Prefecture University and were performed according to the animal ethical guidelines of Osaka Prefecture University. Male C57BL/6J mice (3 months old, SLC Japan, Shizuoka, Japan) were used for A␤40 intracerebroventricular (i.c.v.) injection studies. A triple transgenic mouse model of AD (3ϫTg-AD), harboring human transgenes of amyloid precursor protein (APP SWE ), presenilin-1 (PS1 M146V ), and tau (Tau P301L ) under the control of Thy1.2 promoters (25), and control mice, were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice of both genders were used at 1, 3, 6, and 9 months of age. All animals were maintained on ad libitum ordinary laboratory chow and tap water under a constant 12-h light/12-h dark cycle.
Surgical Procedure-Mice were anesthetized intraperitoneally with sodium pentobarbital (50 mg/kg, intraperitoneally, Abbott) and positioned in a stereotaxic frame (Narishige). The i.c.v. injections of A␤40 aggregates (50 M) incubated with or without 10% GAPDH aggregates were administered at a volume of 2 l using a Hamilton syringe (0.2 l/min) according to the following coordinates: 0 mm anterior to bregma, 0.8 mm lateral from midline, and 2.2 mm ventral from the skull surface. In this study, animals (n ϭ 10) were divided into four treatment groups as follows: 1) control, vehicle, sterilized PBS; 2) aggregated A␤40 (50 M) incubated for 48 h at 37°C in vehicle; 3) aggregated A␤40 (50 M) plus 10% GAPDH aggregates (5 M) in vehicle; and 4) 10% GAPDH aggregates in vehicle. Control and GAPDH siRNA were also injected in 2-l volumes in the same way.
Hematoxylin and Eosin Staining and Immunohistochemistry-Mice were deeply anesthetized with pentobarbital (200 mg/kg, intraperitoneally) and transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde in PBS. The brain was removed and post-fixed in 4% paraformaldehyde in PBS at 4°C overnight and then routinely processed and embedded in paraffin. For morphological analysis, paraffin-embedded brain tissues were cut (5 m thick), deparaffinized, and stained with hematoxylin and eosin. For immunohistochemical analysis, deparaffinized sections were incubated with 100 g/ml proteinase K (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 50 mM Tris-HCl buffer (pH 7.4) for 10 min at 37°C. For detection of A␤, additional incubation with 70% formic acid for 5 min was performed. After three washes with PBS, sections were incubated with 10% goat serum in PBS, followed by incubation overnight at 4°C with mouse anti-A␤ monoclonal antibody (6E10, 1:1000), rabbit anti-GAPDH polyclonal antibody (1:500), rabbit anti-AIF polyclonal antibody (1:500), mouse anti-cytochrome c monoclonal antibody (1:500), and rabbit anti-GFAP polyclonal antibody (1:1000). Sections were then incubated for 1 h at room temperature with Alexa 488-or Alexa 568-conjugated secondary antibodies, goat anti-rabbit IgG (1:1000), goat anti-mouse IgG (1:1000), and DAPI (1:2000) for nuclear staining and then coverslipped in Fluorescein Mounting Medium (Dako). These antibodies were detected using a confocal scanning microscope (model C1si-TE2000-E, Nikon). For quantification of AIF nuclear translocation, cells exhibiting indication of AIF nuclear translocation were counted and expressed as a ratio to the total number of nuclei stained with DAPI. For quantification of GFAP, cytochrome c, A␤ aggregates, and GAPDH aggregates, raw images were first grayed and then transformed into binary images using Scion imaging software (version 4.0.3., Scion Corp.). The number of binary pixels in the square images was measured automatically.
Co-immunoprecipitation-Insoluble fractions prepared from mice hippocampi (0.5 mg/ml, 300 l) were incubated with normal mouse IgG (2 g) and 10 l of 50% protein G-Sepharose slurry (Amersham Biosciences) to pre-clear proteins that bind non-specifically to IgG. After centrifugation (15,000 rpm, 1 min), supernatants were incubated with an anti-A␤ monoclonal antibody (4 g) overnight at 4°C, followed by incubation with protein G-Sepharose for 1 h. The beads were washed four times with 1 ml of modified RIPA buffer containing 20 mM Tris-HCl (pH 7.5), 15 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.1% deoxycholate, 0.1% SDS, 1 mM PMSF, and a protease inhibitor mixture. The beads were added to 40 l of