Distribution of GFP–n-synaptobrevin in the <i>Drosophila</i> brain.

<p><b>A</b>) Optical sections of the brain obtained by confocal microscopy (four brightest sections for each genotype). The fluorescent signal was visualized at a wavelength of λ 488 nm. The scanning sections were 2 µm thick. Microphotographs show the brains of 30-day-old flies. <b>B</b>) Quantitation of relative intensities of GFP–n-synaptobrevin (n-syb-eGFP) in the brain section for transgenic flies with different genotypes: 1.-elav; 2- elav;ΔNAPP; 3- elav;ΔCAPP; 4-elav;APP; 5-elav;APPSw; 6-elav;APP/BACE; 7- elav; APPSw/BACE. The pixel intensity in the selected areas was estimated using Image J software and was normalized to the fluorescent signal of <i>elav</i> genotype. Relative intensities are shown as averages±SEM. p<0.05.</p

Effect of peptides on Aβ accumulation in <i>Drosophila</i> brain.

<p>Immunohistochemistry with 4G8 antibody: <b>A</b>) Distribution of Aβ deposits in <i>Drosophila</i> brain, Bar, 50 µm. <b>B</b>) Effect of peptides on Aβ accumulation in <i>Drosophila</i> brain, Bar, 10 µm.</p

Progressive loss of memory in <i>APP</i>-transgenic-flies.

<p>The Memory Index was calculated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008191#s4" target="_blank">Materials and Methods</a>”. All scores expressed as mean±SEM, (n = 6) where (n) is the number of independent assays. Asterisk (*) indicates control transgenic lines. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer multiple comparison post hoc test. Significance was accepted at p<0.05. Groups of flies showing statistically significant differences from controls in learning index are shown in bold font.</p

Age-dependent neurodegeneration in <i>APP</i> expressing transgenics.

<p>Microphotographs of the brains of 30-day-old flies; Bar, 50 µm. <b>A</b>) Percentage of the area lost in the regions of the cell body and neuropil: The ratio of lost area was calculated by dividing the sum of the vacuole areas from all brain sections by the total area of the brain from all brain sections. We analyzed 6 brains for each genotype, 16–18 sections per brain. Percentage of the area lost is shown as averages±SEM. p<0.05. <b>B</b>) Neurodegeneration in <i>APP</i> flies at day 30. Paraffin slices were stained with hematoxylin and eosin and were examined under bright-field illumination using a Leica DM 2500 microscope at a magnification of 120X.</p

Human <i>APP</i> expression in the brain of transgenic <i>Drosophila</i>.

<p><b>A</b>) Western blot analysis of APP and Aβ: 22C11 (APP-N): Full size human APP was detected by monoclonal antibody 22C11 (APP N terminal-specific); 4G8 (IP): Aβ was immunoprecipitated using anti-Aβ monoclonal 4G8 antibody; elav;APP; and -elav;APP/BACE- genotypes of transgenic strains used for analysis. All blots were scanned and relative intensity of bands was quantified using Image J software. <b>B</b>) Aβ deposits in <i>Drosophila</i> brain: 4G8 (IH): Immunohistochemistry with 4G8 antibody; Arrows indicate areas for magnification. Bar, 50 µm (a, b); Bar, 10 µm (c, d).</p

Progressive loss of learning ability in <i>APP</i>-transgenic flies.

<p>The Learning Index was calculated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008191#s4" target="_blank">Materials and Methods</a>”. All scores are expressed as mean±SEM, (n = 6) where (n) is the number of independent assays. Asterisk (*) indicates control transgenic lines. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer multiple comparison post hoc test. Significance was accepted at p<0.05.</p><p>Groups of flies showing statistically significant differences from controls in learning index are shown in bold font.</p

ApoE-mimetics inhibit neurodegeneration in <i>Drosophila</i> brain.

<p><b>A</b>) Effect of peptides on percentage of the area lost in the regions of the cell body and neuropil: The ratio of lost area was calculated by dividing the sum of the vacuole areas from all brain sections by the total area of the brain from all brain sections. Percentage of the area lost is shown as averages±SEM. p<0.05 Asterisks indicate significant differences from control. <b>B</b>) Effect of peptides on neurodegeneration in <i>APP</i> transgenic flies. Paraffin slices were stained with hematoxylin and eosin and were examined under bright-field illumination using a Leica DM 2500 microscope at a magnification of 120X. Left panel- Bar, 50 µm; Right panels - Bar, 10 µm.</p

Comparison of prevention of neurodegeneration in the brain of <i>APP</i>-transgenic flies by apoE-mimetics after injection and feeding.

<p>The degenerated area was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008191#s4" target="_blank">Materials and Methods</a> at day 30. Six brains were analyzed for each genotype. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer multiple comparison post hoc test. Significance was accepted at p<0.05. Results showing statistically significant decrease of neurodegeneration in comparison with control are shown in bold font. Flies were cultured on standard fly food containing tested peptides, which were added every day at concentrations of: 11.47 µM COG1112, 11.71 µM Antp, and 3.72 µM Antp-SH8.</p><p>0.1 µl injections of 11.74 µM COG133, 11.47 µM COG112, and 11.71 µM Antp into the abdomen where performed every 4 days starting from day 2. All scores expressed as mean±SEM (n = 6).</p

In vivo delivery of peptides into <i>Drosophila</i> brain.

<p>Immunohistochemistry with antibody to biotin: Left panels - Bar, 50 µm; Right panels-Bar, 10 µm. a,b - Antp-COG133 (COG112) - RQIKIWFQNRRMKWKK LRVRLASHLRKLRKRLL. c,d- CF (fragment 142–153 of human growth factor U2AF)-SQMTRQARRLYV, control peptide, which does not cross BBB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008191#pone.0008191-Peng1" target="_blank">[33]</a>; e,f – no peptides added.</p

Effect of COG133 and COG112 on learning and memory in <i>APP</i>-nransgenic flies.

<p>The Learning Index and Memory Index were calculated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008191#s4" target="_blank">Materials and Methods</a>”. All scores expressed as mean±SEM, (n = 6) where (n) is a number of independent assays. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer multiple comparison post hoc test. Significance was accepted at p<0.05. Groups of flies showing statistically significant recovery of learning and memory in comparison with control (Antp) are shown in bold font. 0.1 µl injections of 11.74 µM COG133, 11.47 µM COG112, and 11.71 µM Antp into the abdomen were performed every 4 days starting from day 2.</p