<i>In vitro</i> fibril conversion of Aβ_1–42 is accelerated by curcumin.

<p>Aliquots of fibrillation reactions (10 µM Aβ_1–42 in 10 mM phosphate pH 7.5, 37°C, agitation) were assayed at 0, 60 and 180 min. (A) Native-PAGE separation was interpreted as differential separation of monomeric, oligomeric and insoluble Aβ_1–42. Lower populations of oligomeric species and elevated amounts of insoluble Aβ_1–42 was observed in curcumin containing samples. (B) Transmission electron micrographs obtained at 60 minutes in the absence of curcumin revealed the presence of morphologically disordered aggregates with associated amyloid fibrils, whereas all concentrations of curcumin revealed the presence of amyloid fibrils. (C) At 180 minutes all samples contained extensively fibrillated Aβ_1–42. Scale bars represent 100 nm. (D) p-FTAA fluorescence kinetics assaying formation of prefibrillar Aβ_1–42 aggregates in the presence of vehicle (2% EtOH) and/or 0.0001, 0.001, and 0.01% (w/v) curcumin (0.27–27 µM curcumin). The concentration dependent suppression of the rapid prefibrillar phase, suggests accelerated conversion of Aβ_1–42 to amyloid fibrils in the presence of curcumin. Absence of curcumin is represented with black line, 0.0001, 0.001, and 0.01% (w/v) curcumin is represented in yellow, orange, and red lines respectively.</p

Curcumin accelerates aggregate to fibril conversion in <i>Drosophila</i> brains.

<p>Amyloid fibrillation index as individual spectral shift (A, C, and E) and mean spectral shift with standard deviation (B, D, and F) over time, in presence (orange) or absence (black) of curcumin treatment for Aβ and Tau expressing flies. (A and B) Newly eclosed double insert Aβ_1–42 expressing flies had variable emission spectra, resulting in a wide variation in the 508/612 ratios. Some aggregates with the distinct amyloid feature were also detected (marked with triangles in the graph) in young flies. The double insert Aβ_1–42 expressing flies displays a higher amyloid fibrillation index after ten days of curcumin treatment, indicating a more organized fibril formation due to curcumin ingestion. Untreated flies did not display an increased Aβ-fibrillation index at day 10. At day 20, the overall amyloid fibrillation index had increased, but there were no differences in the amyloid-fibrillation index between untreated and treated flies. (C and D) The Aβ_{1–42 E22G} expressing flies showed a rather high amyloid fibrillation index already in newly eclosed flies. Both treated and untreated flies have higher Aβ-fibrillation index at day 5, but at day 10 the Aβ-fibrillation index was lower, especially for untreated flies. (E and F) The Tau expressing flies showed more red shifted emission spectra than the Aβ genotypes, with a higher contribution from the 612 nm emission (560 nm excitation) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031424#pone.0031424.s003" target="_blank">Figure S3</a>), resulting in a lower amyloid fibrillation index. Note the different scale of the y-axis compared to A–D. A few aggregates with elevated amyloid fibrillation index were detected as marked by triangles in the graph. Overall the trend was the same as for Aβ expressing flies but the statistical analysis revealed that no significant differences were observed for the Tau expressing flies upon curcumin treatment. No spectra could be analyzed in Tau expressing flies at day 0, due to low detection rate and minute aggregates, resulting in low spectral intensities. (ns: non-significant; *: P<0.05, **: P<0.01; ***: P<0.001).</p

Phenotypes of <i>C155-Gal4/UAS-Aβ</i> and <i>C155-Gal4/UAS-tau</i> Alzheimer Disease Models of <i>Drosophila</i> treated and untreated with 0.001% (w/w) of curcumin.

a<p>Defined as total number of beam brakes for 16 flies in the DAM2 system over 24 h. Flies were reared at 29°C and assayed at the same temperature.</p>b<p>Protein deposition load determined by visual inspection with a fluorescence microscope of immunofluorescence of antibody detection, of histological sections of <i>Drosophila</i> heads at day 10 for <i>C155-Gal4/UAS-Aβ_1–40</i>, <i>C155-Gal4/UAS-Aβ_1–42</i>, <i>C155-Gal4/UAS-Aβ_1–42;UAS-Aβ_1–42</i>, <i>C155-Gal4/UAS-tau</i>; and day 5 for <i>C155-Gal4/UAS-Aβ_{1–42 E22G}</i>. At least 20 fly heads were assayed for each genotype. +++, extensive; ++, intermediate; + detectable; −, not detected; n.a., not assayed.</p>c<p>Aggregate deposition load determined by visual inspection with a fluorescence microscope of p-FTAA and immunofluorescence of histological sections of <i>Drosophila</i> heads at day 10 for <i>C155-Gal4/UAS-Aβ_1–40</i>, <i>C155-Gal4/UAS-Aβ_1–42</i>, <i>C155-Gal4/UAS-Aβ_1–42;UAS-Aβ_1–42</i>, <i>C155-Gal4/UAS-tau</i>; and day 5 for <i>C155-Gal4/UAS-Aβ_{1–42 E22G}</i>. At least 20 fly heads were assayed for each genotype. +++, extensive; ++, intermediate; + detectable; −, not detected; n.a., not assayed.</p>d<p>Not all flies showed protein aggregates, given is the load when protein aggregates were detected.</p

Schematic illustration of Aβ aggregation in <i>Drosophila</i>.

<p>The aggregation from soluble Aβ monomer to Aβ oligomers (step 1) and further maturation to Aβ amyloid fibrils (step 2) is dependent on age. The Arctic mutation (E22G) favors the formation of oligomers. In the presence of curcumin, the aggregation into Aβ amyloid fibrils (step 2) is favored.</p

Curcumin affects the brain amyloid deposition histology patterns as a function of <i>Drosophila</i> genotype.

<p>Micrographs of <i>Drosophila</i> brains taken with 100× objective showing fluorescence from cell nuclei by DAPI (blue), amyloid aggregates by p-FTAA (green) and Aβ by αAβ-antibody (red). (A–E) Control flies with and without treatment with curcumin at day 0, day 10, and day 20, showed no antibody or p-FTAA binding species in the brain tissue. (F–J) Double insert Aβ_1–42 expressing flies showed extensive amyloid staining by p-FTAA, with several long extended fibrillar aggregates at all time points. Curcumin treatement accelerated p-FTAA positive (amyloid aggregate) conversion at young age (c.f. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031424#pone-0031424-g003" target="_blank">Figure 3G and H</a>). The amount of detectable aggregates increased with age. DAPI staining from regions with widespread p-FTAA-positive aggregates were decreased. (K–O) Aβ_{1–42 E22G} expressing flies at day 0, day 5, and day 10, showed a spot-like staining from both p-FTAA and the Aβ specific antibody, but a weaker p-FTAA staining was apparent (c.f. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031424#pone-0031424-g003" target="_blank">Fig. 3 I and N</a>). Some irregular nuclei were visible from the DAPI staining. Day 0 and day 5 flies showed protein aggregates detectable with the antibody and to some extent by p-FTAA. The same staining pattern was reveled irrespective of curcumin treatment. Filled arrowheads show p-FTAA positive amyloid aggregates and open arrows indicate αAβ-antibody positive aggregates (diffuse Aβ accumulation). Scale bars in insets represent 20 µm.</p

Curcumin affects Drosophila longevity as a function of genotype.

<p>Survival trajectories of transgenic <i>C155-Gal4 Drosophila</i> for different curcumin treatments indicated by: no curcumin added (black lines), 1, 10, and 100 µg curcumin per g yeast paste represented in yellow, orange, and red lines respectively. (A) Control flies showed a concentration dependent decrease in lifespan upon curcumin treatment. (B) Aβ_1–40 expressing flies showed reduced survival times when fed intermediate and high curcumin concentrations. (C) Single insert Aβ_1–42 expressing flies showed increased survival when treated with low and intermediate curcumin concentrations. (D) Double insert Aβ_1–42 expressing flies showed an increased survival for low and intermediate curcumin concentrations. (E) Aβ_{1–42 E22G} expressing flies showed increased survival for all curcumin concentrations. (F) Survival of Tau expressing flies was unaffected at low and intermediate concentrations of curcumin, but revealed a shortened survival time at the high curcumin concentration. (G) Median survival time of all genotypes with no curcumin added represented in black bars, 1, 10, and 100 µg curcumin per g yeast paste represented in yellow, orange, and red bars respectively. Significance was compared using a paired student's t-test between untreated and treated with curcumin (ns: non-significant; *: P<0.05, **: P<0.01; ***: P<0.001).</p

The amount of Aβ-peptide accumulation in <i>Drosophila</i> brain is not affected by curcumin.

<p>Quantification of Aβ-peptide in aged flies was performed using the Meso Scale Discovery (MSD) immunoassay. Soluble and insoluble Aβ concentrations for untreated flies (black bars) and curcumin treated flies (orange bars). (A) The double insert Aβ_1–42 expressing flies showed an increased signal of Aβ compared to controls at all time points in the soluble fraction. There was no significant difference in the curcumin treated or untreated flies. (B) The double insert Aβ_1–42 expressing flies showed an increased signal, at all time points, of Aβ compared to controls. The insoluble fraction accounted for the majority of Aβ, but there was no significant difference in Aβ concentration between untreated flies and curcumin treated flies. (C) Aβ_{1–42 E22G} expressing flies showed an increased signal of Aβ compared to controls at day 5 and day 10 in the soluble fraction. There was no significant difference in soluble Aβ of curcumin treated flies, when compared to untreated flies. (D) The Aβ_{1–42 E22G} expressing flies showed an increased signal of Aβ when compared to controls at all time points in the insoluble fraction, but there was no significant difference in Aβ concentration between untreated flies and curcumin treated flies. Error bars denote the mean standard deviation as deduced from triplicate samples in three independent experiments. (ns: non-significant; *: P<0.05, **: P<0.01; ***: P<0.001).</p

Direct evidence for Aβ_1–42 binding <i>in vitro</i>.

<p>(A–C) Curcumin degradation at neutral pH (PBS) solution over time assessed by absorbance at 440 nm for solutions containing 0.0001% (yellow), 0.001% (orange), and 0.01% (red) (w/v) curcumin. Solid lines and dotted lines represented in presence and in absence of 10 µM Aβ_1–42 peptide respectively. Black lines represents background absorbance of buffer (dotted line) and 10 µM Aβ_1–42 peptide (solid line) in the absence of curcumin. (D–F) Curcumin absorbance spectra for the corresponding curcumin containing samples as those described in A–C at 0 h, 2 hours and 10 hours of incubation colored in gray scale.</p

Threads may cause damage to axons.

<p>(A) Many APP-positive axonal spheroids (red) can be seen in a representative cross-section from a 27 week old 5xFAD mouse. 6E10-positive threads (green) are often seen co-labeling with APP staining (arrow) at this age. (B) Z-stack images (0. 5μm optical sections) from the spheroid indicated by the arrow in A reveal that a thread lies on the outside of the spheroid rather than inside it. (C) Similarly, a sagittal section showed a 6E10-labeled (green) thread at the tip of an APP-positive (red) spheroid. (D) Sagittal section stained with the aminergic fibre marker, tyrosine hydroxylase (red), and 6E10 (green) shows a thread, extending from a spheroid at the end of an aminergic fibre, that forms a coil about 30μm caudal to the spheroid. Scale bar in A = 50μm; B-C = 10μm and D = 15μm.</p

Threads appear before plaque deposition.

<p>(A) No plaques were found in 8 week old 5xFAD mouse spinal cord, however, 6E10-positive threads can be found (boxed areas) in this sagittal cervical cord section. Only 3 threads were found over the entire length of the cervical cord section. (B) In contrast, at 19 weeks of age numerous plaques were observed in the grey matter at the cervical level in 5xFAD mouse spinal cord (B1), and many threads were observed in the white matter (boxed area in B1 enlarged in B2). Scale bar in A and B1 = 500μm; B2 = 100μm.</p