Effect of IDEQ upon solubility and secondary structure of Aβ aggregates.

<p>(<b>A</b>) 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 <i>t</i>, Aβ trimers. (<b>B</b>) Densitometric quantification of Aβ1-42 obtained from Western blots shown in panel (<b>A</b>). Bars represent the mean ± SEM of total Aβ immunoreactivity in arbitrary units (AU). * p<0.05, Student's <i>t</i> test. (<b>C</b>) 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). (<b>D</b>) 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.</p

Aβ1-42 oligomers formed in the presence of IDEQ are not neurotoxic.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) Analysis of neuronal processes under the conditions as shown in panel (<b>A</b>). 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. (<b>D</b>) 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.</p

Effect of IDEQ and ATP upon the kinetic of Aβ aggregation and seeding.

<p>(<b>A</b>) 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 (<b>•</b>). 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>B</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. (<b>C</b>) 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 (□). (<b>D</b>) 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.</p

IDEQ does not modify insulin conformation.

<p>(<b>A</b>) <i>Circular dichroism (CD) spectra</i> 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>B</b>) Same samples as in panel (<b>A</b>) after incubation for 24 h at 25°C. Insulin alone (solid black line), insulin with IDEQ (solid gray line) and IDEQ alone (dotted line). (<b>C</b>) 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.</p

Time-course, amount and pH-dependence of the formation of complexes between Aβ1-42 and IDEwt or IDEQ.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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 (<b>A</b>), (<b>C</b>) and (<b>D</b>), IDEwt or IDEQ-AβSCxs are indicated by arrowheads.</p