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 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 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

Autophagosome-lysosome fusion in the cortex of untreated and OLE-fed wt and Tg mice.

<p>(A) Merge of cathepsin B (green) and p62 (red) immunoreactivity in 3.5-month-old wt and Tg mice. Co-localization between cathepsin B and p62 staining was detected as bright yellow puncta in small-sized lysosomes in the cortex of untreated wt mice and OLE-fed Tg and wt mice (n = 6/group). Scale bar = 25 µm. Inset: high magnification of a p62 and cathepsin B positive neuron. Scale bar = 14 µm. (B) Western blotting analysis of cathepsin B and p62 levels in the cortex of 3.5-month-old wt and Tg mice; wt = pool of untreated and OLE-fed wt mice (n = 10); Tg = untreated and OLE-fed Tg mice (N = 6/group). Both cathepsin B and p62 levels were significantly increased in OLE-fed Tg mice, as compared to untreated Tg mice. Data are representative of four experiments and are normalized for β-actin and reported as mean values ± S.E.M. *P<0.05, ** P<0.01. (C) Single and double fluorescent immunohistochemistry with cathepsin B (green) and p62 (red) Abs in the cortex of untreated and OLE-fed Tg mice. In the untreated Tg mice, bright cathepsin B immunoreactivity occurred in enlarged lysosomal compartments (arrows), p62 immunoreactivity was light and no co-localization between cathepsin B and p62 was found. Inset: high magnification of a cell with cathepsin B-positive giant lysosomes. In the OLE-fed Tg mice, a bright cathepsin B immunoreactivity appeared in small-sized lysosomes, p62 immunoreactivity was greater than in untreated Tg mice and a significant co-localization between cathepsin B and p62 was evident. (n = 6). Scale bars = 25 µm applies for single cathepsin B staining and 10 µm applies to the inset and high magnification images.</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

Astrocyte and microglia reaction in the brain of untreated and OLE-fed TgCRND8 mice.

<p>(A) reconstruction of representative photomicrographs of GFAP immunoreactivity in the cortex and hippocampus. Hypertrophic astrocytes with long and thick branches were detected in the brain of untreated Tg mice. In the OLE-fed animals the astrocyte reaction was considerably milder. Insets: high magnification of GFAP-positive astrocytes (n = 4–5/group). Scale bars: 500 µm for all reconstructed images and 25 µm for all insets (B) Iba1 immunopositive microglial cells in the cortex. Note the presence of microglia with enlarged cell bodies, thickened and retracted processes (n = 4–5/group) in the cortex of OLE-fed 6-month-old Tg mice. Scale bar: 25 µm applies to all images. (C) TBARS in cortical homogenates of 3.5-month-old mice showing that lipid peroxidation was not significantly reduced by OLE treatment. *P<0.05. Data are reported as mean values ± S.E.M. (n = 3–4 mice/group). Each sample was analyzed in two replicates.</p

OLE restores cognitive performance in 3.5–6 month-old TgCRND8 mice.

<p>(A) Step down test: one-way ANOVA plus Bonferroni’s post comparison test shows a statistically significant increase in the mean retention latencies in untreated and OLE-treated wt and in OLE-treated Tg mice, as compared to their respective training latencies (§P<0.001). Untreated Tg mice do not show significant differences between training and retention latencies (P>0.05). The retention latencies of untreated Tg mice significantly differ from the retention latencies of all the other groups (#P<0.001). (B) ORT: in the T2 trial the discrimination index of untreated Tg mice significantly differ from the discrimination index of all other groups (*P<0.05). The dotted line indicates the chance level performance; Data are reported as mean values ± S.E.M. Number of animals: n = 12/group.</p