OLE reduces amyloid deposition and extends the life-span of CL2006 transgenic <i>C. elegans</i> strain.

<p>Egg-synchronized CL2006 worms were placed at 16 °C on fresh NGM plates seeded with OP50 <i>E.coli</i> and, at L2 larval stage, were fed with vehicle or with 50 µM OLE for 48 h. (<b>A</b>) At 120 h of age, corresponding to day 1 of adult age, worms were stained with X-34 dye. The staining of amyloid plaques on whole-mount and fixed samples was visualized at short wavelength excitation. Scale bar 20 µm. Arrows in red indicate amyloid deposits. (<b>B</b>) Amyloid deposits in the anterior area of worms fed with vehicle (n = 20) and OLE (n = 20), quantified by counting the number of X-34 positive spots. **p<0.01 vs. CL2006 worms fed with vehicle (Student’s t test). (<b>C–D</b>) Kaplan-Meier survival curves of (<b>C</b>) CL2006 and (<b>D</b>) CL802 worms fed with vehicle or with 50 µM OLE at L2 larval stage. The number of paralyzed worms (considered dead) was scored 48 h after treatment, at L4 larval stage (day 0 in graph) and every consecutive day, until all worms were dead. Survival is expressed as a percentage of the initial population. Plots are representative of three independent experiments (n = 30 worms/group).</p

Effect of OLE on Aβ-induced paralysis in CL2006 transgenic <i>C. elegans</i> strain.

<p>(<b>A</b>) Diagram illustrating the paralysis assay showing when the drug was administered and when the paralysis assay was scored. (<b>B–C</b>) Dose-response effect of OLE on the paralysis induced by Aβ expression in CL2006 and CL802 transgenic worms. Egg-synchronized worms were placed at 16 °C on fresh NGM plates seeded with OP50 <i>E. coli</i> and, at L2 (<b>B</b>) or L3 (<b>C</b>) stage, were fed with OLE (12.5–500 µM). The number of paralyzed worms was scored 48 h or 24 h after treatment (at L4 larval stage) for L2- and L3-treated worms, respectively. Data are shown as percentage±SE of paralyzed worms to vehicle treated ones (n = 100 worms/group, 3 independent assays). (<b>D</b>) Percentage of paralyzed worms fed with OLE. CL2006, CL802 and N2 worms, cultured as above, were fed 50 µM OLE at L1 or L2 and 500 µM at L3. Tetracycline at 50 µM was administered at L3 as positive control. The number of paralyzed worms was scored at L4 larval stage. Data are shown as percentage±SD of paralyzed worms to vehicle treated ones (n = 100 worms/group, 3 independent assays). °° p<0.01 vs. CL802, **p< 0.01 vs CL2006 worms fed with vehicle (One-way ANOVA test), and +p<0.01 vs. CL2006 worms fed with 50 µM OLE at L2 (Student’s t test).</p

Oleuropein Aglycone Protects Transgenic <em>C. elegans</em> Strains Expressing Aβ42 by Reducing Plaque Load and Motor Deficit

<div><p>The presence of amyloid aggregates of the 42 amino acid peptide of amyloid beta (Aβ42) in the brain is the characteristic feature of Alzheimer’s disease (AD). Amyloid beta (Aβ deposition is also found in muscle fibers of individuals affected by inclusion body myositis (sIBM), a rare muscular degenerative disease affecting people over 50. Both conditions are presently lacking an effective therapeutic treatment. There is increasing evidence to suggest that natural polyphenols may prevent the formation of toxic amyloid aggregates; this applies also to oleuropein aglycone (OLE), the most abundant polyphenol in extra virgin olive oil, previously shown to hinder amylin and Aβ aggregation. Here we evaluated the ability of OLE to interfere with Aβ proteotoxicity <i>in vivo</i> by using the transgenic CL2006 and CL4176 strains of <i>Caenorhabditis elegans</i>, simplified models of AD and of sIBM, which express human Aβ in the cytoplasm of body wall muscle cells. OLE-fed CL2006 worms displayed reduced Aβ plaque deposition, less abundant toxic Aβ oligomers, remarkably decreased paralysis and increased lifespan with respect to untreated animals. A protective effect was also observed in CL4176 worms but only when OLE was administered before the induction of the Aβ transgene expression. These effects were specific, dose-related, and not mediated by the known polyphenolic anti-oxidant activity, suggesting that, in this model organism, OLE interferes with the Aβ aggregation skipping the appearance of toxic species, as already shown <i>in vitro</i> for Aβ42.</p> </div

Effect of OLE on Aβ oligomer production.

<p>Representative dot blot of (<b>A</b>) total Aβ (WO2) and (<b>C</b>) Aβ oligomers (A11) in CL2006 transgenic worms fed with vehicle or with 50 µM OLE at L2 larval stage. Equal amounts of proteins from worm lysates (5 µg) were spotted in triplicate. Total proteins on the blotted membranes were stained using a 0.1% Red Ponceau solution and were used to normalize the immuno-specific signal for protein loading. The mean volume of the Aβ-reactive and Red Ponceau-dyed spots was determined using the Progenesis SameSpots software (Nonlinear Dynamics, UK). Immunoreactivity of WO2 (<b>B</b>) or A11 (<b>D</b>), from three independent experiments (n = 9), was expressed as the mean volume of the immunoreactive band/volume of Ponceau-dyed proteins±SD.*<i>p</i><0.05 vs. vehicle (Student’s t-test).</p

Effect of OLE on oxidative stress in CL2006 transgenic <i>C. elegans</i> strain.

<p>Egg-synchronized CL2006 transgenic worms were placed at 16 °C on <i>E. coli</i> for 48 h and then fed, at L2 larval stage, with vehicle or with 50 µM OLE for 48 h (100 µl/plate). Tetracycline and NAC were used as positive controls for antioxidant activity. Worms were then collected in 1.6 ml 1% Tween 20 in PBS and the NBT assay was done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058893#s2" target="_blank">Materials and Methods</a>. Results are calculated as absorbance units/mg of protein (n = 100 worms/group, three independent experiments) and expressed as % of control, i.e. the percentage of superoxide produced by drug-treated CL2006 worms, considering 100% that produced by vehicle treated ones.**<i>p</i><0.01 vs. vehicle-treated CL2006 worms, according to One-way ANOVA and Bonferroni’s post test analysis.</p

OLE increases Beclin 1 and LC3 in the cortex of wt and TgCRND8 mice.

<p>Representative images of Beclin 1 (A) and LC3 (C) immunoreactivity showing an intense bright and punctate Beclin 1 staining in the soma, perikarya and dendrites of neurons and strong and bright LC3 puncta in the neuronal cell bodies and processes of neurons in the somatosensory/parietal cortex of Tg mice and, to a lesser extent, in the wt mice fed with OLE, as compared to age-matched untreated Tg and wt mice (n = 5/group). Scale bars = 50 µm applies for the low magnification images and 20 µm applies for the high magnification images of untreated and OLE-fed 6-month-old Tg mice. (B) and (D) Western blotting analysis of Beclin 1 (B) and LC3 (D) levels in cortical tissue, exemplified for mice of 3.5 months of age, normalized for β-actin, (n = 6–7/group). LC3 levels are expressed as LC3-II/LC3-I levels. In the cortex of OLE-fed animals Beclin 1 levels show a trend towards an increase in the wt mice and in the OLE-fed Tg mice Beclin 1 and LC3 levels were significantly increased respect to age-matched untreated wt and Tg mice. (**P<0.01). Data are reported as mean values ± S.E.M.</p

OLE activates autophagy in N2a murine neuroblastoma cells.

<p>The cells were exposed to 90 µM (A) or 50 µM (C) OLE for increasing time periods. (B) and (D): the cells were exposed for 6 h to increasing OLE concentrations. Cells were lysed and analyzed by western blotting as described. This is a representative experiment out of three that gave qualitatively identical results.</p

OLE reduces plaque burden in the cortex and hippocampus of TgCRND8 mice.

<p>(A) Representative photomicrographs of Aβ42 immunopositive deposits. (n = 5/group, six sections from each mouse). Insets: high magnification images of representative plaques. Scale bars = 500 µm applies to all reconstructed images and 20 µm to all magnified images. (B) Quantitative analysis of total plaque area and plaque number in untreated and OLE-fed Tg mice (n = 6 for 3.5 and 6 months Tg mice). (C) ELISA: cortical levels of SDS- and formic acid-soluble Aβ40 and Aβ42 peptides in OLE-fed and untreated Tg mice. Both Aβ40 and Aβ42 levels were significantly decreased in OLE-fed Tg versus age-matched untreated Tg (n<i> = </i>5/group) mice. *P<0.05, **P<0.01 and ***P<0.001. Data are reported as mean values ± S.E.M. mo = month-old.</p

Antibodies employed in the study.

<p>Legend: aa, amino acid; WB, western blot; IHC, immunohistochemistry, ND, not done.</p

OLE modifies Aβ plaque load and morphology in the brains of TgCRND8 mice.

<p>Representative photomicrographs of Thioflavin S histochemistry (green) (n = 4/group) and OC immunolabeling (red) (n = 5/group) of amyloid plaques in the cortex of untreated and OLE-fed Tg mice. In the OLE-fed Tg mice of 6 months of age several radiating plaques with ribbon-like/diffuse core and fluffy deposits (arrow) are present. Arrowhead indicates dense core amyloid plaques. Scale bars = 25 µm.</p