Figure 1.
Time-course, amount and pH-dependence of the formation of complexes between Aβ1-42 and IDEwt or IDEQ.
(A) Western blots with anti-Aβ 6E10 showing the ∼120 kDa band corresponding to IDE-AβSCx (IDE-Aβ stable complex) as a function of the incubation time. Top panel, IDEQ; lower panel, IDEwt. Both PVDF membranes were developed simultaneously with a STORM 860 scanner. Below each Western blot, the same membranes stained with Coomassie blue, show IDEwt or IDEQ loading. (B) Densitometric data from Western blots for IDEQ (◯) and IDEwt (▴) were fitted to a single exponential equation using Graph Pad Prism v.4 software. Points represent the mean ± SEM from two independent experiments in duplicate. (C) IDEQ-AβSCx formation is partially competed by pre-incubation for 1 h with insulin at the indicated molar excess before the addition of Aβ1-42. Data are expressed as the percentage of the remaining Aβ-positive band at ∼120 kDa, in arbitrary units, as a function of insulin concentration. Each point represents the mean ± SEM of two independent experiments in duplicate. Inset: a representative Western blot of IDEQ-AβSCx developed with 6E10. (D) Densitometry of IDEQ-AβSCx at the indicated range of pH as determined by Western blot with anti-Aβ. Bars represent the mean ± SEM of three separate experiments. Inset: top, representative Western blot with anti-Aβ of IDEQ-AβSCx; bottom, Coomassie blue of IDEQ loaded in each lane. In panels (A), (C) and (D), IDEwt or IDEQ-AβSCxs are indicated by arrowheads.
Figure 2.
IDEQ does not modify insulin conformation.
(A) Circular dichroism (CD) spectra of 10 μM insulin (ins.) in working buffer (solid black line), insulin with IDEQ at 1∶100 molar ratio, enzyme:insulin (solid gray line) and IDEQ alone (dotted line) with no prior incubation. (B) Same samples as in panel (A) after incubation for 24 h at 25°C. Insulin alone (solid black line), insulin with IDEQ (solid gray line) and IDEQ alone (dotted line). (C) Western blot with anti-phospho-Akt and anti-total Akt of U-87 cell lysates. Cells were exposed for 30 min with insulin alone, insulin previously co-incubated with IDEwt or IDEQ, as indicated. Wortmannin (wort) was incubated at 10 nM for 30 min before treatments.
Figure 3.
Time-course of Aβ self-assembly assessed by DLS and TEM in presence or absence of IDEQ.
(A) Dynamic light scattering (DLS) time-course of Aβ1-42 incubated alone or in the presence of IDEQ, as indicated. White, gray and black areas correspond to “dead time”, 48 h and 160 h of incubation, respectively. Arrows indicate Aβ species with very high hydrodynamic diameter (DH) (≧1000 nm). Aβ1-42 was incubated at a 10-molar excess over IDEQ. Results are representative of three independent experiments. Data were plotted using Sigma Plot 5.3 software to graphically show the DH (in a logarithmic scale) and the percentage of scattering intensity (I.Scatt) (in a linear scale). (B) Transmission electron microscopy (TEM) analysis of: Aβ1-42 alone showing typical amyloid fibrils (top left); Aβ1-42 in the presence of IDEQ (middle left) showing coalescent annular species of approximately 5–10 nm and the absence of amyloid fibrils. Arrow indicates a large aggregate. Inset, annuli indicated by small arrows; IDEQ alone (bottom left) forming annular structures of 5–10 nm (inset, large arrows) and thin short rods (inset, thin arrows); Aβ1-42 incubated with IDEwt inhibited with EDTA (top right), showing no amyloid fibril formation. Inset, arrows depict 5–10 nm annuli and rods. Electron micrographs were obtained after 6.5 days of incubation. Samples incubated for 30 days revealed typical amyloid fibrils for Aβ1-42 alone (middle right) in contrast to a homogeneous population of small annuli of 5–10 nm in the presence of IDEQ (bottom right). In all TEM experiments, molar ratios were 1∶10 (enzyme:Aβ). Bars in left panel and top and middle right panel = 100 nm, in insets, bars = 20 nm. In bottom right, bar = 30 nm.
Figure 4.
Effect of IDEQ and ATP upon the kinetic of Aβ aggregation and seeding.
(A) Turbidity profiles of Aβ1-42 at 15 μM alone in working buffer (▴) or in the presence of IDEQ at the indicated molar ratios (IDEQ:Aβ), from top to bottom: 1∶200 (□), 1∶100 (Δ), 1∶10 (◯) and 1∶10 containing 0.5 mM ATP (•). Light scattering at 340 nm was measured every 30 min using a TECAN GENios multi-well reader (for clarity, only the points every other 90 min are shown). The bracket encloses the curves obtained after co-incubation of Aβ1-42 with IDEQ at the indicated conditions. Results are expressed as mean ± S.E.M. of at least two independent experiments in duplicate. (B) Representative TEM images of samples at steady state of Aβ1-42 alone (top) or with IDEQ at 1∶10 molar ratio in the presence of ATP (bottom). Bars = 100 nm. (C) Time course of Aβ1-42 aggregation alone (▴), in the presence of seeds previously formed with IDEQ (◯), or after the addition of pure Aβ1-42 seeds (□). (D) Kinetics of aggregation of Aβ1-42 after the addition of IDEQ (◯) or the same volume of working buffer (▴) to Aβ1-42 after 48 h of self-assembly, as indicated by the arrow.
Figure 5.
Effect of IDEQ upon solubility and secondary structure of Aβ aggregates.
(A) samples containing Aβ1-42 before incubation (“dead time”) and after incubation for 5 days with or without IDEQ were centrifuged at 3,000× g for 5 min and supernatants and pellets analyzed by Western blots with anti-Aβ 6E10 and 4G8. Arrowheads indicate: H, high molecular mass oligomers, T, Aβ tetramers and t, Aβ trimers. (B) Densitometric quantification of Aβ1-42 obtained from Western blots shown in panel (A). Bars represent the mean ± SEM of total Aβ immunoreactivity in arbitrary units (AU). * p<0.05, Student's t test. (C) Far UV-CD spectra recorded at “dead time” of Aβ1-42 (solid black line), IDEQ alone (dotted line) and Aβ1-42 co-incubated with IDEQ (solid gray line) at a 1∶300 molar ratio (IDEQ:Aβ). (D) Far UV-spectra recorded after 6 days of incubation of Aβ1-42 alone or Aβ1-42 with IDEQ at 1∶300 molar ratio. Samples were centrifuged as described above and supernatants analyzed. IDEQ alone, dotted line; Aβ1-42 alone, solid black line; Aβ1-42 co-incubated with IDEQ, solid gray line.
Figure 6.
Aβ1-42 oligomers formed in the presence of IDEQ are not neurotoxic.
(A) Representative AFM images showing the size and morphology of Aβ1-42 neurotoxic species. Left, Aβ1-42 incubated alone for 4 days. Approximately spherical species of ∼20–30 nm are indicated by arrowheads. Rods and short protofibrils are depicted by arrows. Inset: a larger Aβ1-42 protofibril is shown. Right; Aβ1-42 incubated in the presence of IDEQ at a 1∶10 molar ratio (IDEQ: Aβ) showing larger aggregates of 50–60 nm (arrowheads) and rods with lengths of ∼100–120 nm (arrows). (B) Representative immunofluorescence of primary differentiated neurons exposed to vehicle, Aβ1-42 alone or Aβ1-42 pre-incubated with IDEQ from top to bottom, as indicated. White arrows point at neuronal processes. Bars = 30 μM. (C) Analysis of neuronal processes under the conditions as shown in panel (A). Bars represent the mean ± SEM of processes' lengths as measured from the centre of the neuronal body * p<0.01, one-way ANOVA, Tukey post-hoc test. (D) Viability of mature primary neurons after the indicated treatments as assessed by MTT reduction. * p<0.05, one-way ANOVA, Tukey post-hoc test. Results are shown for three independent experiments.