Fly Stocks and Antibodies
Transgenic fly lines carrying the human Aβ42 was established in the background of the Canton-S w1118
) genotype as described in 
. The elav-GAL4c155
line was outcrossed with the isoCJ1
flies for 5 generations. The X-linked dnc
allele, which was crossed into a background containing the iso1CJ
autosomes, was a kind gift from Dr. T. Tully (Cold Spring Harbor Laboratory). A control cross to iso1CJ
also was used. Other fly stocks and antibodies were obtained from: Drs. W. M. Saxton (UAS-mito-GFP, University of California, Santa Cruz), K. E. Zinsmaier (miro[Sd32]
, The University of Arizona), L. Luo (UAS-CD8::GFP;;OK107, Stanford University), M. B. Feany (UAS-α synuclein, Harvard Medical School), the Bloomington Drosophila
Stock Center (Indiana University) (elav-GAL4c155
, gmr-GAL4, Cha-GAL4, UAS-tub-GFP, UAS-syt-GFP, rut
, PKA-R2 [EP2162]
and PKA-R2 [EY11150]
), the VDRC stock center (UAS-milton RNAi flies (v41507 (labeled as #2) and v41508 (labeled as #1)), UAS-PKA-C1 RNAi (v6993) and UAS-PKA-R2 RNAi (v39436)) 
, T. L. Schwarz (anti-Drosophila
milton, Harvard Medical School), and D. Kalderon (anti-PKA-C1 and anti-PKA-R2, Columbia University). A control cross to w1118
from Bloomington Stock Center or w1118
from VDRC was used for these flies. Anti-RRxpS/T (Cell Signaling, Beverly, MA) and anti-tubulin (Sigma, St. Louis, MO) were purchased.
GFP Analysis in Fly Brains
Fly brains were dissected in cold PBS, fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences), and then placed under vacuum in PBS containing 4% paraformaldehyde and 0.25% Triton X-100. The fluorescence intensity in the mushroom body regions was analyzed using a confocal microscope (Carl Zeiss LSM 510) and quantified using NIH image.
Genomic DNA Extraction and Quantitative Real Time PCR Analysis
Fly brains were dissected in cold PBS and frozen on dry ice, and genomic DNA was extracted. 20 brains were homogenated in 100 mM Tris-HCl pH 7.5, 100 mM EDTA, 100 mM NaCl, and 0.5% SDS, and incubate at 65°C for 30 min. Samples were treated with 1.5 M potassium acetate and 4 M LiCl, and incubated for 65°C for 30 min, and centrifuged. Supernatant was treated was phenol/chloroform, added isoprophanol, and centrifuged. Precipitated gemonic DNA was rinsed with 70% ethanol and subjected to quantitative real time-PCR (Applied Biosystems). The average threshold cycle value (Ct) was calculated from five replicates per sample. Levels of Co I, Co III and CytB DNA were standardized relative to that of rp49. Relative expression values were determined by the deltaCt method according to quantitative PCR Analysis User Bulletin (Applied Biosystems). Primers were designed using NIH primer blast as follows: Co I, CTGGAATTGCTCATGGTGGA (forward) and CTCCCGCTGGGTCAAAAA (reverse); Co III, CCCGCTATTGAATTAGGAGCA (forward) and ATTCCGTGGAATCCTGTTGC (reverse); CytB, TGAGGTGGATTTGCTGTTGA (forward) and TGGTTGAATATGGGCAGGTG (reverse); rp49, GCTAAGCTGTCGCACAAATG (forward) and GTTCGATCCGTAACCGATGT (reverse).
ATP, PKA, and cAMP Assays
ATP contents in dissected brains without eye pigments were analyzed using ATP Bioluminescence Assay Kit CLSII (Roche, Mannheim). PKA activity in dissected brains without eye pigments was measured with MESACUP Protein Kinase Assay Kit (MBL, Woburn, MA) in the presence or absence of 2 µM cAMP. cAMP levels was measured with cAMP-screen system (Applied Biosystems, Foster City, CA). ATP, PKA and cAMP levels were calculated by standard curves and normalized by protein levels.
Transmission Electron Microscopy
Probosces were removed from decapitated heads, which were then immersion-fixed overnight in 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS. Samples were post-fixed 1 hr in ferrocyanide-reduced osmium tetroxide (1% osmium tetroxide and 1.5% potassium ferrocyanide in distilled water). Fixation was followed by dehydration in a graded ethanol series and infiltration with Epon-Araldite resin (2 hr in 50% resin in acetone and 24 hr in 100% resin) using constant rotation. After transferring the samples to flat-bottom BEEM capsules with fresh resin, the samples were polymerized overnight at 60°C. Cured blocks containing fly heads were examined with a dissection microscope and heads with a suitable orientation (posterior oriented flat to the block surface) were selected for thin sectioning. Semi thin sections stained with toluidine blue were examined by light microscopy to localize the mushroom body region. Thin sections (120 nm) of entire heads were collected on nickel grids (100 mesh, Veco-EMS). Thin sections were stained for 5 minutes in lead citrate stain. Sections were examined and micrographs collected using a Hitachi H700T TEM.
Fly brains were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), treated with 25 µg/ml proteinase K for 30 min, and incubated with In Situ Cell Death Detection Kit, Fluorescein (Roche, Mannheim) for 1 hr at 37°C. The brains were analyzed using a confocal microscope (Carl Zeiss LSM 510).
RNA Extraction and Quantitative Real Time PCR Analysis
For each sample, 30–40 flies were collected and frozen. Heads were mechanically isolated, and total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol with an additional centrifugation step (11,000×g for 10 min) to remove cuticle membranes prior to the addition of chloroform. Total RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen), and the resulting cDNA was used as a template for PCR on a 7500 fast real time PCR system (Applied Biosystems). The average threshold cycle value (Ct) was calculated from five replicates per sample. Expression of milton was standardized relative to actin. Relative expression values were determined by the deltaCt method according to quantitative PCR Analysis User Bulletin (Applied Biosystems). Primers were designed using NIH primer blast as follows: milton, CAGGATCAGCTGAAGCAACA (forward) and ACACGCTACCTCCCATTGTC (reverse); and actin5C, TGCACCGCAAGTGCTTCTAA G (forward) and TGCTGCACTCCAAACTTCCA (reverse).
Dissected brains were homogenized in Tris-glycine sample buffer (Invitrogen) and centrifuged at 13,000 rpm for 10 min, and the supernatants were separated on 6% or 10% Tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% nonfat dry milk (Nestlé) and blotted with the primary antibody (anti-Drosophila milton (a gift from Dr. T. L. Schwarz), anti-PKA-C1 (a gift from Dr. D. Kalderon), anti-PKA-R2 (a gift from Dr. D. Kalderon), anti-RRxpS/T (Cell Signaling), or anti-tubulin (Sigma)), incubated with appropriate secondary antibody and developed using ECL plus Western Blotting Detection Reagents (GE Healthcare).
Climbing assay was performed as previously described 
. Approximately 25 flies were placed in an empty plastic vial. The vial was gently tapped to knock the flies to the bottom, and the number of flies at the top, middle, or bottom of the vial was scored after 10 seconds. Experiments were repeated more than three times, and a representative result was shown.
Fly brains were dissected in cold PBS, fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences), and then placed under vacuum in PBS containing 4% paraformaldehyde and 0.25% Triton X-100. After permeabilization with PBS containing 2% Triton X-100, the brains were stained with rabbit polyclonal anti-PKA-C1 antibody (a gift from Dr. D. Kalderon) followed by detection with biotin-XX goat anti-mouse IgG and streptavidin-Texas Red conjugate (Molecular Probes). The brains were analyzed using a confocal microscope (Carl Zeiss LSM 510).
Sequential Extraction and Western Blotting of Aβ42
For sequential extractions of Aβ42, fly heads were homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl) containing 1% SDS. Lysates were centrifuged at 100,000×g for 1 h, and supernatants were collected (SDS-soluble fraction). SDS-insoluble pellets were further homogenized in 70% formic acid (Sigma) followed by centrifugation at 13,000 rpm for 20 min, and the supernatants were collected (formic acid fraction). Formic acid was evaporated by SpeedVac (Savant, SC100), and protein was resuspended in dimethyl sulfoxide (Sigma). Protein extracts were separated on 10–20% Tris-Tricine gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were boiled in phosphate-buffered saline (PBS) for 3 min, blocked with 5% nonfat dry milk, blotted with the 6E10 antibody (Signet), incubated with appropriate secondary antibody and developed using ECL plus Western Blotting Detection Reagents (GE Healthcare).
Thioflavin S Staining
For thioflavin S (TS) staining, the dissected brains were permeabilized and incubated in 50% EtOH containing 0.1% TS (Sigma) overnight. After washing in 50% EtOH and PBS, the brains were analyzed using a confocal microscope. The numbers of TS-positive deposits were quantified from four hemispheres from three flies per genotype. The fluorescence intensity in Kenyon cell regions was analyzed using a confocal microscope (Carl Zeiss LSM 510) and quantified using NIH image.
Quantification of Neurodegeneration
For the analysis of neurodegeneration in Kenyon cell region, heads were fixed in 4% paraformaldehyde, processed to embed in paraffin blocks, and sectioned at a thickness of 6 µm. Sections were placed on slides, stained with hematoxylin and eosin (Vector Laboratories), and examined by bright field microscopy. To quantify neurodegeneration, images of the sections were captured, and the areas of the vacuoles were measured using NIH Image.
Detection of Phosphorylated dCREB by Immunoprecipitation
and the transgenic hs-dCREB2d line are from Dr. J. C.- P. Yin. Fly heads were homogenized in RIPA buffer (50 mM Tris·HCl, pH 8.0/0.5% sodium deoxycholate/1% Triton X-100/150 mM NaCl) containing 1% SDS, centrifuged, and supernatant was collected. Protein extracts were diluted to 1
10 with RIPA buffer and immunoprecipitated with the anti-RRxpS/T antibody (Cell Signaling, Beverly, MA), separated on 10% Tris-Glycine gel (Invitrogen), and blotted with the anti-dCREB antibody (a gift from Dr. J. C.- P. Yin).