Roles of neuronal and peripheral CREBs in energy stores.

<p>(A) Neuronal overexpression of a dominant-negative form of CREB (DN-CREB) caused lower stored glycogen and lipid levels in flies. Glycogen (left) or lipid (middle) content in bodies from control flies (<i>elav</i>-GAL4 driver only, control) or flies expressing DN-CREB in neurons from the <i>elav</i>-GAL4 (DN-CREB) driver. Glycogen and lipid levels were normalized to protein levels and expressed as ratios to the control level (mean±SD, n = 4, *p<0.05, Student's t-test). Body size (right) of flies with DN-CREB expression in neurons was indistinguishable from that of the control. Measurements of mesothorax size are shown as ratios to the control size (mean±SD, n = 8). Two independent transgenic lines (DN-CREB#1 and DN-CREB#2) gave similar results. (B) Reduction in CREB activity in flies following DN-CREB expression in the fat body. CRE-Luciferase reporter protein was measured using anti-luciferase antibody in Western blots of body extracts from control flies (<i>to</i>-GAL4 driver only, control) or flies expressing DN-CREB in the fat body from the <i>to</i>-GAL4 driver (DN-CREB) (top panel). Blots were stripped and reprobed with anti-tubulin antibodies as a protein loading control (bottom panel). Signal intensities were quantified and are shown as ratios to control signals (mean±SD, n = 5; *p<0.05, Student's t-test). (C) Overexpression of DN-CREB in the fat body caused lower stored glycogen and higher lipid contents. Glycogen (left) or lipid (middle) content in the bodies of control flies (<i>to</i>-GAL4 driver only, control) or flies expressing DN-CREB in neurons from the <i>elav</i>-GAL4 (DN-CREB) driver. Glycogen and lipid levels are expressed as ratios to the control levels, n = 6, *p<0.05, Student's t-test). Body size (right) of flies expressing DN-CREB in the fat body was indistinguishable from that of the control. Measurements of mesothorax size are shown as ratios to the control values (mean±SD, n = 8).</p

Roles of neuronal and peripheral CREBs in food intake.

<p>The ingestion of dye was quantified after feeding 1-week-old male flies for 6 h (left) or 24 h (right). The absorbance of ingested dye was measured and the results are shown as ratios to the control value (mean±SD, *p<0.05, Student's t-test). (A) Flies with DN-CREB expression in neurons. (B) Flies with DN-CREB expression in the fat body.</p

Reduction in CRE-mediated transcription by knock-down of AKHR in the fat body.

<p>Total RNA was extracted from flies and subjected to qRT-PCR. (A, C, and E) AKHR mRNA level, (B, D, F and G) CRE-luciferase mRNA levels. (mean±SD, n = 5, *p<0.05, Student's t-test).</p

MOESM3 of Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Additional file 3: Fig. S3. Locomotor activity in AppNL-G-F/NL-G-F and AppNL/NL mice during the pre-shock period in the contextual fear conditioning task. The distance travelled during the pre-shock period (3-min period just prior to the first footshock) in conditioning was compared among genotypes at both 6–9 (a and b) and 15–18 (c and d) months of age. Representative images of movement tracks during the pre-shock period in each genotype at 6–9 (a) and 15–18 (c) months of age were shown. At 6–9 months of age, AppNL-G-F/NL-G-F mice exhibited a slight decrease in distance travelled during the pre-shock period in comparison with WT mice (b). At 15–18 months of age, AppNL/NL mice exhibited a significant decrease in distance travelled during the pre-shock period in comparison with WT mice (d). Locomotor activity in AppNL-G-F/NL-G-F mice was also slightly decreased in comparison with WT mice. 6–9 month-old; n = 6 WT (B6 J), n = 6 AppNL/NL, n = 9 AppNL-G-F/NL-G-F. 15–18 month-old; n = 8 WT (B6 J), n = 7 AppNL/NL, n = 7 AppNL-G-F/NL-G-F. *p < 0.05 versus WT (B6J)

MOESM2 of Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Additional file 2: Fig. S2. Locomotor activity of AppNL-G-F/NL-G-F and AppNL/NL mice during the first and second trials in the elevated plus maze task. The distance travelled during the 10-min test of the first and second trials in the elevated plus maze task was compared among genotypes at both 6–9 (a–d) and 15–18 (e–h) months of age. Representative images of movement tracks during the first and second trials for each genotype at 6–9 (a and c) and 15–18 (e and g) months of age were shown (closed arms are indicated by shaded areas). At 6–9 months of age, AppNL-G-F/NL-G-F mice exhibited slight increases in distance travelled during the first (b) and second (d) trials in comparison with WT mice. By contrast, locomotor activity in AppNL/NL mice was comparable with WT mice in the two trials. At 15–18 months of age, AppNL-G-F/NL-G-F mice exhibited a slight increase in movement compared to WT mice during the first (f) and second (g) trials. AppNL/NL mice moved at similar levels compared with WT mice in the two trials. 6–9 month-old; n = 8 WT (B6J), n = 8 AppNL/NL, n = 8 AppNL-G-F/NL-G-F. 15–18 month-old; n = 12 WT (B6J), n = 10 AppNL/NL, n = 11 AppNL-G-F/NL-G-F. †p < 0.05 versus AppNL/NL

Normal brain structure and expression and distribution of proteins related to neuron and glia in Thr⁶⁶⁸Ala mutant mice.

<div><p>(A) Nissl-stained hippocampal sections show no difference between wild-type (T/T) and Thr⁶⁶⁸Ala mutation homozygotes (A/A).</p> <p>Scale bars represent 500 µm.</p> <p>(B) Immunohistochemical analysis in CA1 hippocampal region of aged (>12 mo-old) mice.</p> <p>Immunostaining for GFAP (astroglial marker), synaptophysin (presynaptic marker), and MAP-2 (neuronal dendritic marker) in hippocampal CA1 region of wild-type (T/T) and Thr⁶⁶⁸Ala mutant (A/A) mice are shown.</p> <p>S.o., stratum oriens; Py, pyramidal cell; Rad, stratum radiatum. Scale bars represent 50 µm.</p> <p>(C) Western blot analysis of APP, X11L, MAP2, synaptophysin, PSD95 (postsynaptic marker), and GFAP from the brains of 12 mo-old wild-type (T/T) and Thr⁶⁶⁸Ala mutant (A/A) mice.</p></div

Generation of Thr⁶⁶⁸Ala Knock-in Mutant Mice.

<div><p>(A) The targeting vector (a1), a partial map of the APP gene (a2), the resultant targeted allele (a3), and the knock-in allele after Cre recombination (a4) are illustrated.</p> <p>Filled boxes denote coding sequences of exons 17 and 18.</p> <p>Shaded parts in exon18 correspond to the 3′ non-coding region.</p> <p>A substitution is represented by a dot in exon 18.</p> <p>B, <i>BamHI</i>; X, <i>XhoI</i>.</p> <p>Probes for Southern blot analysis for screening of targeted ES clones are indicated with small bars in (a2–4).</p> <p>PCR primers (E1 and E2) for genotyping of mice are indicated with small arrows in (a2 and a4).</p> <p>(B) Verified sequences from wild-type (T/T), and Thr⁶⁶⁸Ala mutation homozygotes (A/A).</p> <p>The Thr⁶⁶⁸ flanking genomic region was amplified using mouse tail DNA as the template by PCR and sequenced.</p> <p>(C) Southern blot analysis for targeted ES cells and PCR analysis for genotyping wild-type and knock-in mouse lines.</p> <p>DNA from G418-selected ES cells was digested with <i>XhoI</i> and analyzed by Southern blotting with a 3′ external probe.</p> <p>The 13-kb and 15-kb fragments represent wild-type and targeted alleles, respectively. PCR fragments of 200-bp and 298-bp represent wild-type and knock-in alleles, respectively.</p> <p>(D) Immunoblot analysis of mouse whole brain.</p> <p>APP was immunoprecipitated with anti-pan APP polyclonal antibody followed by immunoblotting with anti-pan APP antibody UT-421 or anti-phospho-Thr⁶⁶⁸-specific antibody UT-33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000051#pone.0000051-Iijima1" target="_blank">[7]</a>.</p> <p>APP from homozygotes has no immunoreactivity with UT-33 although the APP expression levels detected by UT-421 were indistinguishable from those of wild-type mice.</p> <p>Mature APP (mAPP; <i>N</i>- and <i>O</i>-glycosylated form), immature APP (imAPP; <i>N</i>-glycosylated form), and phosphorylated APP (pAPP) are indicated with arrows.</p></div

Aging-dependent phosphorylation of APP and APP CTFs and quantification of CTFs in wild-type and Thr⁶⁶⁸Ala mutant mice brain.

<div><p>(A) APP phosphorylation state in brains of post-natal day 0 (P0), young adult (2-month), and aged adult (12-month) mice.</p> <p>The upper panel was probed with anti-pan-APP C-terminal antibody G369, and the lower panel was probed with an anti-phospho-threonine-⁶⁶⁸-specific antibody.</p> <p>W, wild-type mouse; M, Thr⁶⁶⁸Ala mutant mouse.</p> <p>(B) APP carboxyl-terminal fragments (APP CTFs) in wild-type (W) and mutant (M) mouse brain.</p> <p>C99 and C89 are products resulting from cleavage of APP by BACE, while C83 results from cleavage of APP by ADAM-10/-17.</p> <p>PhosphoC99 (pC99), phosphoC89 (pC89), and phosphoC83 (pC83) are all mono-phosphorylated at Thr⁶⁶⁸, and these peptides are numbered here according to standard APP₆₉₅ nomenclature.</p> <p>(C) Expression levels of CTFα and CTFβ in middle aged wild-type and Thr⁶⁶⁸A mutant mouse brain.</p> <p>Various species of CTF are schematically represented at the left and indicated at right with bars.</p> <p>Samples were electrophoresed after treatment with either buffer or λ phosphatase (λ PPase).</p> <p>The amounts were normalized to unity for wild-type mice (1.0).</p> <p>The bars indicate means±S.D. (N.S.; n = 6).</p></div

Hemi-brains from wild-type (upper panel of six blots, labeled “Wild-type”, far left) or mutant (lower panel of six blots; labeled “Mutant T⁶⁶⁸A”, far left) 5-month old mice were used for fractionation on iodixanol density-gradients as described (4).

<p>Equal aliquots (according to volume) were analyzed by immunoblotting with antibodies as specified: anti-early endosome antigen 1 (Transduction laboratories, EEA1, top panel), anti-<i>cis</i>-Golgi matrix protein (Transduction laboratories, GM130, second panel), anti-protein disulfide isomerase (Stressgen, PDI, a marker for the endoplasmic reticulum, third panel), anti-post synaptic density 95 (Transduction laboratories, PSD95, a synaptic membrane marker, fourth panel), anti-pan-APP (G369, fifth panel) and anti-phospho-threonine⁶⁶⁸ APP (Cell Signaling, bottom panel).</p

Additional file 10: of Integrated biology approach reveals molecular and pathological interactions among Alzheimer’s Aβ42, Tau, TREM2, and TYROBP in Drosophila models

Table S9 Module membership from weighted gene co-expression network analysis for ROSMAP gene expression data. (XLSX 456 kb