Figure 1.
RI-OR2 and RI-OR2-TAT inhibit the aggregation of Aβ42.
(A) ThT data for Aβ42 incubated with and without these inhibitors: ‘con’ is buffer only; ‘Aβ’ is Aβ42 incubated at 25 µM for 48 h, with no inhibitor; the dark grey bars are Aβ42 incubated for 48 h with RI-OR2 at 1–25∶25, 2–12.5∶25, 3–6.75∶25, 4–2.5∶25 µM concentration of inhibitor:Aβ; and the black bars are Aβ42 incubated with RI-OR2-TAT at these same molar ratios. (B) Effects of the inhibitors on detection of multimeric Aβ42 by immunoassay. The data for Aβ42 alone, incubated at 12.5 µM, are shown on the left (pale bars), and for Aβ42 incubated with 25∶12.5, 12.5∶12.5, 6.75∶12.5 and 2.5∶12.5 µmolar concentrations of RI-OR2-TAT:Aβ on the right (black bars). In each case, the consecutive bars are for 0, 4, 8, 24 and 48 h incubations. Statistics: for both (A) and (B), *denotes p<0.05 for treated sample versus untreated control, and **denotes p<0.01. (C) AFM images of a 24 h incubation of Aβ42 (25 µM) in the presence and absence (Control) of RI-OR2-TAT (12.5 µM). Scale bar is to the right.
Figure 2.
Binding of RI-OR2-TAT to immobilized Aβ42 fibrils, as shown by surface plasmon resonance (SPR) spectroscopy.
The peptide was injected in solution at three different concentrations, for 5 mins at a flow rate of 30 µl/min. The upper line shows data for 6 µM RI-OR2-TAT, the middle line for 3 µM RI-OR2-TAT and the lower line for 1 µM RI-OR2-TAT. The non-specific binding obtained from the reference surface has been automatically subtracted from all data. Fitted curves are shown in black.
Figure 3.
RI-OR2-TAT inhibits the toxic effects of Aβ42 on cells.
(A) The black bars show data for the viability of SHSY-5Y neuroblastoma cells, as measured by LDH assay, following exposure to 12.5, 25, 50, 100 and 200 µM RI-OR2-TAT alone for 24 h. The light grey ‘Con’ bar shows data for cells maintained under the same conditions, but without RI-OR2-TAT, and the dark grey bar shows data for lysed cells. (B) Shows the ability of RI-OR2-TAT to protect against Aβ42-mediated toxicity. The black bars are LDH assay data for cells grown in the presence of 5 µM Aβ42 plus 0.1, 0.5, 1, 5 or 10 µM RI-OR2-TAT for 24 h. The ‘0’ bar shows data for cells grown in the presence of Aβ42 alone and the bars labelled ‘Con’ and ‘Lysed cells’ are the same as for (A). For both (A) and (B) *indicates p<0.05.
Figure 4.
Flu-RI-OR2-TAT enters cultured SHSY-5Y cells and crosses the blood brain barrier of APP/PS1 transgenic mice.
(A) Fluorescent microscope image of SHSY-5Y cells exposed to 1 µM RI-OR2-TAT for 10 mins. (B) Corresponding image for cells exposed to Flu-RI-OR2, also at 1 µM, but for 1 h. (C) Fluoresence images showing the detection of Flu-RI-OR2-TAT in sections of brain tissue following i.p. injection in 17-month old APP/PS1 transgenic mice. (D) Shows the same sections as in (C) but with detection using AlexaFluor 555 labelled antibody against either (on the left) Aβ or (on the right) activated microglia/Iba 1. (E) Shows merged images of (C) and (D) above. Flu-RI-OR2-TAT is seen to be co-localised with Aβ and with activated microglial cells.
Figure 5.
RI-OR2-TAT reduces the β-amyloid plaque load and levels of Aβ soluble oligomers in the brains of APP/PS1 transgenic mice.
Representative images show amyloid deposits in the cortex region of 10 months old APP/PS1 mouse brains as shown by β-amyloid immunostaining in animals treated with (A) 0.9% saline or (B) 100 nmol/kg RI-OR2-TAT in 0.9% saline. (C) Quantitative analysis shows a decrease in mean plaque load in the cortex of APP/PS1 mice treated with RI-OR2-TAT compared to animals treated with saline. (D) Levels of soluble Aß oligomers in these brains. Values represent mean ± SEM of 4 animals per group, where *** p<0.0001; ** p<0.01, unpaired Student t-test.
Figure 6.
RI-OR2-TAT reduces the brain load of microglia and oxidative damage and stimulates neurogenesis in APP/PS1 transgenic mice.
Representative images of activated microglia in the cortical region of APP/PS1 mouse brains as shown by Iba1 immunostaining in animals treated with (A) 0.9% saline or (B) 100 nmol/kg RI-OR2-TAT in 0.9% saline; (C) quantitative analysis shows a decrease in mean microglial load in APP/PS1 mice treated with RI-OR2-TAT; detection of 8 oxo-guanine in the brains of animals treated with (D) 0.9% saline or (E) 100 nmol/kg RI-OR2-TAT; (F) quantitative analysis shows a decrease in mean 8 oxo-guanine staining in APP/PS1 mice treated with RI-OR2-TAT; detection of immature neurons as stained for doublecortin in the dentate gyrus of APP/PS1 mice treated with (G) 0.9% saline or (H) 100 nmol/kg RI-OR2-TAT; (I) quantitative analysis shows an increase in mean doublecortin staining in APP/PS1 mice treated with RI-OR2-TAT. Values represent mean ± SEM of 4 animals per group, where *** p<0.0001, unpaired Student t-test.