Figure 1.
Diagram showing the locations of spin labels in Aβ(1–40), and the structures of the HO-4160 and K01-162 fluorene compounds.
Top panel:. the spin labels were targeted to either position 2 (MTS nitroxide spin label) or position 26 (TOAC nitroxide spin label) of Aβ. Position 26 lies within a putative hairpin loop connecting the terminal domains of the peptide, while position 2 is found within the N-terminal domain. Bottom panel: the structure of the SLF compound HO-4160, which is a derivative of the active fluorene K01-162 described in [6]. The core fluorene ring structure is shown in red.
Figure 2.
SLF HO-4160 protects against AβO toxicity in cultured neurons.
Plotted are the viability values for neuroblastoma MC65 cells with conditional APP-C99 expression as a function of compound concentration. The protective effect against AβO toxicity exceeds that of the previously described base compound, K01-162 (insert).
Table 1.
Potency of SLF compounds [15] against Aβ toxicity as determined by the MTT assay.
Figure 3.
SLF HO-4160 greatly diminishes the intracellular population of oligomeric Aβ Shown are Western blots of extracts from the MC65 human neuroblastoma cell line that conditionally expresses C99, a 99-residue carboxyl terminal fragment of APP in the absence of tetracycline (−TC).
C99 is subsequently cleaved by cellular γ-secretase to generate Aβ. TP17, an inactive tricyclic pyrone, serves as a negative control [31], while vitamin E (Vit E), a potent antioxidant that was shown to also block AβO formation in MC65 cells [30], serves as a positive control. p8 is an unresolved band that could be an Aβ homodimer, or a heterodimer of Aβ and APPΔ31 [31], a caspase cleavage product involving residues just downstream from the Aβ origin on APP. The presence of this band does not correlate with MC65 cell death [31]. Blotting was carried out using the Aβ antibody 6E10 (upper panel) and the loading control (lower panel) was probed using an antibody directed against β-actin. The blot shown is representative of 3 replicates.
Figure 4.
SLF blocks the appearance of oligomeric Aβ species within cultured cells.
MC65 cells were treated with the indicated compounds (1 µM) immediately after the removal of the transgene suppressor tetracycline (−TC; panels B–D) to induce AβO accumulation. At 24 hours, cells were fixed, immunofluorescently stained with the oligomer-specific antibody A11 (red) and counterstained with the nuclear dye DAPI (blue). As shown in panel (D), the cytoprotective SLF HO-4160, as well as the antioxidant vitamin E (Vit E) attenuate the accumulation of intracellular AβO (red fluorescent puncta).
Figure 5.
Clearance of AβO aggregates observed by AFM imaging and ThT binding assay.
Small oligomers used in this study are formed within 60 minutes (scale bar 10 nm). The direct visualization of oligomers by using AFM revealed the formation of AβO aggregates. (A) 50 µM of Aβ after 24 hours of incubation at room temperature (diameter 5–10 nm). B) 50 µM of Aβ and 50 µM of SLF after 24 hours of incubation at room temperature. (C) Staining for beta-rich assemblies by the amyloid dye thioflavin T for incubations of Aβ with and without SLF HO-4160 as described in Methods. Data are the averages from 3 separate experiments with the error bars representing the SEM.
Figure 6.
EPR examination indicates the motional freedom of SLF is reduced by interaction with AβO.
AβO imparts a broad component (arrow) into the EPR spectrum of HO-4160, reflecting a population of SLF with a slower correlation time.
Figure 7.
Spectral broadening due to immobilization and dipolar interaction in assemblies of Aβ.
(A) Changes in the correlation time of Aβ(26TOAC) with increased incubation time. To mainly observe the effects on dynamics, the spins in the Aβ preparation were diluted with wild-type peptide (Aβ(1–40)), so that the sample contained only 25% Aβ(26TOAC). (B) Comparisons of EPR spectra of 80 µM AβO preparations containing either 25% (black line) or 100% (red line) Aβ(26TOAC). Both spectra were acquired after 1 hour at room temperature.
Figure 8.
Fluorenes bind AβO and destabilize highly organized secondary structure.
EPR spectra of SLF (10 µM) with single-cysteine mutant Aβ(1–40) oligomers (40 µM) show that motional freedom of the mixture increased following a 2-hour incubation at room temperature (red) compared with 0 hours (black). A) and B) 26 AβO: spin labeled at 26th residue C) and D) 2 AβO: spin labeled at 2nd residue.
Figure 9.
The comparison of EPR spectra from Aβ alone, an actual mixture of SLF and Aβ, and the mathematical sum of the individual EPR signals from SLF and Aβ.
Data were taken for samples containing AβO spin labeled at the 2nd residue (A) and for samples containing AβO spin labeled at the 26th residue (B).
Figure 10.
Circular dichroism analysis of secondary structural changes in Aβ.
The time zero (t = 0) trace was obtained by scanning Aβ immediately after introduction into buffer. Inclusion of SLF had no significant effect on the t = 0 spectrum (not shown). Additional traces were obtained at 1, 2, 4, 6 and 24 hours. For both the control and +SLF samples, the t = 2, 4, and 6 hour traces are not shown, as these curves changed in a stepwise manner between the t = 1 and t = 24 hour time points. See Methods for additional details.
Figure 11.
The EPR spectra of BMPO adducts with hydroxyl (A) and superoxide (B) radicals (generated via ironsulfate/H2O2 and horseradish peroxidase/H2O2, respectively) are shown in green.
The spectra in the presence of SLF are shown in red, reflecting a decrease in the amount of BMPO adduct formed. Based on the difference in the spectral intensities of the generated BMPO-adduct, SLF HO-4160 is able to scavenge approximately 80% and 25% of the superoxide and hydroxyl radicals, respectively.
Figure 12.
Cultured neuronal cells treated with SLF HO-4160 decrease production of hydrogen peroxide.
MC65 cells were cultured in the presence (TC+) or absence (TC−) of tetracycline, a transgene suppressor of the APP fragment C99. In the absence of tetracycline, cells express the C99 fragment which is then further cleaved to form Aβ. Treatment with either the HO-4160 compound (+4160, 0.3 µM) or the antioxidant vitamin E (+Vit E, 100 µM) reduces the extracellular accumulation of hydrogen peroxide in cells expressing APP-C99. Error bars represent the SEM for N = 3. *p<0.05 TC− compared to both + Vit E and +4160.
Figure 13.
Model of action of SLFs on AβO assemblies.
The illustration highlights the bifunctional properties of the SLF, including its ability to block the formation of nm particles and disrupt small oligomers, as well as its antioxidant activity. The ability of SLF to disrupt fibrils or more mature fibrillar oligomers [32] is undetermined.