Figure 1.
Human APP expression in the brain of transgenic Drosophila.
A) 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) Aβ deposits in Drosophila brain: 4G8 (IH): Immunohistochemistry with 4G8 antibody; Arrows indicate areas for magnification. Bar, 50 µm (a, b); Bar, 10 µm (c, d).
Figure 2.
Distribution of GFP–n-synaptobrevin in the Drosophila brain.
A) 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) 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 elav genotype. Relative intensities are shown as averages±SEM. p<0.05.
Figure 3.
Age-dependent neurodegeneration in APP expressing transgenics.
Microphotographs of the brains of 30-day-old flies; Bar, 50 µm. A) 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) Neurodegeneration in APP 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.
Table 1.
Progressive loss of learning ability in APP-transgenic flies.
Table 2.
Progressive loss of memory in APP-transgenic-flies.
Figure 4.
In vivo delivery of peptides into Drosophila brain.
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 [33]; e,f – no peptides added.
Figure 5.
ApoE-mimetics inhibit neurodegeneration in Drosophila brain.
A) 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) Effect of peptides on neurodegeneration in APP 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.
Table 3.
Effect of COG133 and COG112 on learning and memory in APP-nransgenic flies.
Figure 6.
Effect of peptides on Aβ accumulation in Drosophila brain.
Immunohistochemistry with 4G8 antibody: A) Distribution of Aβ deposits in Drosophila brain, Bar, 50 µm. B) Effect of peptides on Aβ accumulation in Drosophila brain, Bar, 10 µm.
Table 4.
Comparison of prevention of neurodegeneration in the brain of APP-transgenic flies by apoE-mimetics after injection and feeding.