Figure 9

<p>A–D: Uptake of fluorescein labeled Aβ42 (F-Aβ42) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, by differentiated PC12 cells following 30 min incubation. (A) F-Aβ42 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B show limited co-localization of F-Aβ42 and AF633-Trf. (D) A magnified portion of image C (enclosed in the white rectangle) to indicate the lack of co-localization of both fluorophores. I–II: Histograms of fluorescence intensity in differentiated PC12 cells treated with (I) F-Aβ42; and (II) AF633-Trf, at 37°C and 4°C.</p

Cellular uptake of a fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf) in wild type mouse brain slices after 30 min incubation.

<p>A–B: Uptake of F-Aβ40 by a subpopulation of (A) cortical neurons (20×) and; (B) hippocampal neurons (20×). C–I: Effect of temperature on the uptake of F-Aβ40 and AF633-Trf by the pyramidal neurons (63× and 3× optical zoom). C–F: Uptake of F-Aβ40 and AF633-Trf at 37°C (C) F-Aβ40 uptake; (D) AF633-Trf uptake; (E) Superimposition of images C and D; (F) Limited co-localization of F-Aβ40 and AF633-Trf, indicated by white masked areas, was found only around the edges of pyramidal neurons. G–I: Uptake of F-Aβ40 and AF633-Trf at 4°C (G) F-Aβ40 uptake; (H) Inhibition of AF633-Trf internalization; (I) Superimposition of images G and H.</p

Figure 5

<p>A–C: Co-localization of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf) in PC12 cells following: (A) 15 min; (B) 45 min; and (C) 60 min incubation. White masked areas indicate the extent of co-localization of F-Aβ40 and AF633-Trf. D: Cellular internalization of F-Aβ40 and AF633-Trf established by the XY, XZ, and YZ projections of differentiated PC12 cells treated with the fluorophores for 60 min. Optical sections (planes 1–45) were obtained from the coverslip bottom to the cell surface with a 0.6 µm Z-step interval.</p

Figure 10

<p>A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, in rat primary hippocampal (RPH) neurons following 30 min incubation at 37°C. (A) F-Aβ40 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B; (D) Overlay of fluorescence images on the DIC image of RPH neurons. E–G: Uptake of F-Aβ40 and AF633-Trf in RPH neurons at 4°C. (E) Uptake of F-Aβ40; (F) No significant neuronal uptake of AF633-Trf at 4°C; (G) Superimposition of images D and E on the DIC image of RPH neurons; H–J: Uptake of F-Aβ40 and AF633-Trf in RPH neurons treated with 10 mM Sodium Azide and 50 mM 2-deoxy glucose, agents that are known to deplete cellular ATP. (H) Uptake of F-Aβ40; (I) No significant cellular uptake of AF633-Trf was observed; (J) Superimposition of images H and I on the DIC image of neurons.</p

Figure 2

<p>(I) Effect of donor concentration and temperature on the uptake of ¹²⁵I-Aβ40 in wild type (WT) mouse brain slices. (II) Effect of endocytotic inhibitor dansyl cadaverine on the uptake of ¹²⁵I-Aβ40 (450 ng/ml) in WT mouse brain slices. (III) Histograms of fluorescence intensity in differentiated PC12 cells exposed to various concentrations of F-Aβ40. (A) Untreated cells; (B) Cells incubated with 0.65 µM F-Aβ40; (C) Cells incubated with 1.3 µM F-Aβ40; (D) Cells incubated with 3.2 µM F-Aβ40; (E) Cells incubated with 3.2 µM F-Aβ40+32 µM unlabeled Aβ40.</p

Figure 11

<p>A–D: F-Aβ40 uptake into the acidic compartments of bovine brain microvascular endothelial (BBME) cells labeled by Lysotracker Red® (60×). (A) Uptake of F-Aβ40 (B) Uptake of Lysotracker Red®; (C) Superimposition of images A and B; (D) A magnified portion of image C (enclosed in the white rectangle) to show co-localization of both fluorophores. I–II: Histograms of fluorescence intensity in BBME cells treated with (I) F-Aβ40: (A) Untreated control, (B) at 4°C, (C) at 37°C; and (II) AF633-Trf: (A) Untreated control, (B) at 4°C, (C) at 37°C.</p

Figure 4

<p>A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 633 labeled transferrin (AF633-Trf), clathrin-mediated endocytosis marker, by differentiated PC12 cells following 30 min incubation. (A) F-Aβ40 uptake; (B) Uptake of AF633-Trf; (C) Superimposition of images A and B; (D) Sparse co-localization of F-Aβ40 and AF633-Trf as indicated by the white masked areas. E–G: Uptake of F-Aβ40 and AF633-Trf in differentiated PC12 cells subjected conditions that inhibit clathrin mediated endocytosis (hypotonic shock for 5 min followed by incubation with potassium free salt solution for 30 min). (E) Uptake of F-Aβ40; (F) Substantial reduction in AF633-Trf; (G). Superimposition of images E and F on the differential interference contrast (DIC) image to show the condition of the cells.</p

Figure 6

<p>A–C: Accumulation of fluorescein labeled Aβ40 (F-Aβ40) and Dil labeled low density lipoprotein (Dil-LDL) in the late endosomes of differentiated PC12 cells following a 15 min treatment. (A) F-Aβ40 uptake; (B) Uptake of Dil-LDL; (C) Superimposition of images A and B indicate only a partial co-localization, shown by the white masked areas. D: Co-localization of F-Aβ40 and Dil-LDL in differentiated PC12 cells following a 30 min treatment.</p

Fibril formation of Aβ derivatives.

<p>(A) Thioflavin T signal versus time of amyloid β peptides. Peptide samples, at 20 µM and containing thioflavin T, were excited at 430 nm and the emissions at 485 nm were monitored over 2.5 days at 37°C with periodic shaking. For each peptide, n = 18. (B) Thio-T fluorescence representing the fibril formation kinetics of various Aβ40 derivatives fitted to the Finke-Watzsky 2-step aggregation model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008813#pone.0008813-Morris1" target="_blank">[36]</a>. The observed data has been indicated by various symbols. The predicted data obtained through curve-fitting is presented as solid lines. (C) TEM confirmation of the presence of amyloid fibrils formed by the corresponding amyloid β peptide described in panel A (a: Aβ40 H13,14G; b: Aβ40 H13G; c: Aβ40; d: Aβ40 H13R; e: Aβ40 R5G, Y10F, H13R). The sample was collected at the end of the fibril formation experiment and applied to a grid and negatively stained with uranyl acetate. Scale bar equals 1 µm.</p

In vitro binding of F-Aβ40 and F-Aβ40 H13,14G in neurons of 4 month old WT brain slices.

<p>(A) Cortex incubated with F-Aβ40. (B) Cortex incubated with F-Aβ40 H13,14G. (C) Hippocampus incubated with F-Aβ40 (D) Hippocampus incubated with F-Aβ40 H13,14G. Scale bar equals 50 µm. (E) Mean fluorescent intensity of cortical and hippocampal neurons from brain slices incubated with F-Aβ40 or F-Aβ40 H13,14G. Analysis of Variance (ANOVA) [F(1,8) = 33.73; p<0.001] followed by Bonferroni post-hoc multiple comparisons: Cortex - **p<0.01; Hippocampus - *p<0.05.</p