Aβ(25-35) aggregation on DOPC lipid bilayer.

<p>3×3 µm2 (a-c), and 5×5 µm2 (d-f), topographic AFM images (1024×1024px²) showing the time evolution of Aβ(25-35) on DOPC lipid bilayer. AFM measurements were performed in tapping mode and in PBS with a continuous scanning of the same area after the addition of the peptide. Over time, Aβ(25-35) forms layered aggregates (LA) on the bilayer surface, bilayer defect areas increase in size and Aβ(25-35) gradually covers the disrupted areas with disordered aggregates (DA). (g) Graph showing the increase in surface area covered by LA (blue line) and the evolution of lipid bilayer over time (red line). Data were qualitatively fitted by rational and sigmoidal functions for the DOPC bilayer (θDOPC) and the LA domains (θP), respectively. Fitting curves act as a guide-to-the-eye. (h) Height distribution histograms measured at t = 30 min (black), 1 h (red), 2 h (green), 3 h (blue), 4 h (cyan) and 5 h (magenta). The squared dashed-line in (e) indicates the area where statistical analyses on 5×5 µm2 AFM images were performed so as to keep the same scan size amongst images.</p

Aggregation of Aβ(25-35) on mica.

<p>Topographic AFM images of Aβ(25-35) aggregates grown on mica. Images were acquired in tapping mode and in PBS. (a) After 6 hours, Aβ(25-35) forms a smooth homogeneous layer without any well-formed aggregates (3×3 µm², 2459×2459px²). (b) On a smaller scale the peptide tends to organize into a texture with some discernible protrusions (white ellipse) (500×500 nm², 1228×1228px²).</p

Aβ(25-35) aggregation on DOPC/DHA lipid bilayer.

<p>(a-c) 5×2.2 µm² (1024×431px²) topographic AFM images of Aβ(25-35) aggregation on DOPC/DHA lipid bilayers. (d) Graph showing the increasing surface area covered by LA (blue line) and the lipid bilayer evolution over time (red line). LA structures were grown within the first 30 min of peptide deposition while the lipid surface area decreased slowly from 96% to 91%. Data were qualitatively fitted by rational and sigmoidal functions for the DOPC/DHA bilayer (Θ_DOPC/DHA) and the LA domains (Θ_P), respectively. Fitting curves act as guide-to-the-eye. (e) Height distribution histograms measured at t = 0 min (black), 30 min (red), 1 h (green), 1 h45 min (blue), 2 h (cyan) and 2 h20 min (magenta).</p

DOPC and DOPC/DHA lipid bilayers.

<p>(a) Height AFM images of DOPC (2.5×2.5 µm² 1024×1024px²) and DOPC/DHA (b) (2.5×2.5 512×512px²) lipid bilayers performed in tapping mode and in PBS. The bilayers cover 92% (DOPC) and 96% (DOPC/DHA) of the mica surface. On DOPC lipid particles collected at the border or within defects are due to incomplete bilayer formation or to defective rinsing.</p

Aβ(25-35) high resolution analysis.

<p>Topographic AFM images (a, c) and corresponding phase images (b, d) performed in PBS on the same area (360×360 nm², 1024×1024 px²) of Aβ(25-35) LA domains on the DOPC bilayer. Images were acquired with an average tip-sample force of 170pN by scanning from left to right (a, b) and from right to left (c, d). Topographies clearly show both globular aggregates (circled) and annular structures (squared). The complex LA aggregate (arrow) in the bottom right-hand corner and the globular aggregate in the top right-hand corner (insets) are perturbed by the tip movement. (e, f) Height (black) and phase (red) line profiles of globular (e) and annular structures (f) measured along the white dashed line in Fig. 6a and 6c. (g,h) Height images of a highly dense globular structure region (286×286 nm², 574×574px²) acquired with two different tip-sample forces. At 220 pN (g), the globular structures are not perturbed, while at 234 pN (h) they are mechanically removed leaving the underlying annular structures. (i, l) AFM height images (770×770 nm², 633×633 px²) of LA on two different regions of the DOPC/DHA bilayer. After 1 h45 min of peptide deposition (i) the LA presents a linear organization highlighted by grey fibres. After 2 h20 min (l) the LA forms a structured layer where linear organization is less visible though still distinguishable. In some locations, it is organized into annular structures (red circles) similar in dimension but more sporadic and of different nature compared to the ones observed on DOPC.</p

Western blot analysis of Kv 2.1 and Kv4.2 subunit expression in cerebral cortex and hippocampus.

<p>Representative immunoblot of cerebral cortex and hippocampus enriched membrane proteins (50 µg/lane) from (Ctr), Aβ_25–35, Aβ_25–35+SP and SP treated rats. Protein markers are shown at right (in kDa). The immunoreactive signals for <b>a</b>) Kv2.1 and <b>b</b>) Kv4.2 were quantified and normalized against β-actin and expressed as a percentage of control (CTR). Data represent mean (±SEM) from 3 independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Ctr value).</p

Immunofluorescence analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Upper panel. Representative immunofluorescence photomicrographs showing Kv1.4 expression in <b>a</b>) hippocampus and <b>b</b>) frontal cortex after memory tests in the four experimental treatments: Control (Saline), Aβ_25–35-i.c.v. treated rats (Abeta), Aβ_25–35-i.c.v. and SP-i.p. treated rats (Abeta+SP), SP-i.p. treated rats (SP). Brain sections were labeled with the neuronal marker NeuN (green) and with the anti Kv1.4 antibody (red). As shown by the merge channel all neurons are Kv1.4 positive. Note the diffuse increase in Kv1.4 fluorescence intensity in the Abeta group and the decrease in the Abeta+SP group compared to the Control. Scale bar: a) 20 µm; b) 60 µm. Lower panel. Histograms showing image analysis performed on neuronal cytoplasm (first row) and the surrounding neuropil (second row). The indexes used were: total fluorescence intensity, vesicles diameters, and vesicles fluorescence intensity. Data represent means (±S.E.M.) obtained from three independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Saline; #p<0.05, ##p<0.01 versus Aβ_25–35treatment).</p

SP reduced Aβ25–35-induced overexpression of Kv1.4 subunit in rat hippocampal neurons.

<p><b>a</b>) Example of Western blot obtained from hippocampal cultures exposed to 20 µM Aβ_25–35 (Aβ alone or in the presence of SP (100 nM) and analyzed 48 h later using a polyclonal antibody against Kv1.4 subunit. The same blots were stripped and reprobed with an antibody against β-actin as internal control (lower panels). Quantitative analysis is depicted below the blots and was determined by band densitometry analysis considering the values found in CTR cells as 100. Data represent means (±S.E.M.) obtained from 4 independent experiments run in duplicate. (**p<0.001 versus CTR, #p<0.05 versus Aβ_25–35 treatment). <b>b</b>) Representative immunofluorescence photomicrographs showing Kv1.4 expression in primary hippocampal cultures. Note the increase in immunofluorescence in the Aβ_25–35 neurons, as compared to control neurons, reversed by SP treatment. Images were obtained from three independent experiments. Scale bar: 20 µm.</p

Western blot analysis of Kv1.4 subunit expression in hippocampus and cerebral cortex.

<p>Representative immunoblot of (<b>a</b>) hippocampus and (<b>b</b>) cerebral cortex enriched membrane proteins (50 µg/lane) from (Ctr), Aβ_25–35, Aβ_25–35+SP and SP treated rats. Protein markers are shown at right (in kDa). The immunoreactive signals at 97 and 110 kDa were quantified and normalized against β-actin and expressed as a percentage of the control (Ctr). Data represent mean (±SEM) from 5 independent experiments. Statistically significant differences were calculated by one-way analysis of variance (ANOVA) for repeated measures followed by Tukey's test for multiple comparisons (**p<0.01 versus Ctr value; #p<0.05 versus Aβ_25–35 treatment).</p

Neuroprotective effects of SP on memory impairments induced by intracerebroventricular injection of Aβ25–35.

<p>(a) Timeline and experimental design. All animals received an infusion (i.c.v.) of Aβ_25–35 (2 µg/µl; 10 µL injection volume) or its vehicle (PBS 10 µL injection volume) and daily treated (7 days) with SP (50 µg/ml/Kg, i.p.) or its vehicle (saline solution 0.9%, i.p.). On the 31^st day after surgery rats were given a daily training session of 4 trials for 3 consecutive days (days 31^st–33^rd). On the 34^th day after surgery the retention of the spatial training was assessed during a 1 min probe trial. On the 35^th day after surgery rats were given a daily training session of 5 trials for 4 consecutive days (days 35^th–38^th). (b) Mean (±S.E.M.) distance traveled to the escape platform on 4 trials of 3 consecutive days of acquisition learning sessions. (c) Time spent (mean ±S.E.M.) during the 1-minute probe trial in the target quadrant and (d) illustrative paths of all animals for the probe test session. (e) Mean (±S.E.M.) distance traveled to the escape platform on 4 trials of 4 consecutive days of the reversal learning sessions (the hidden platform were relocated in a new position each day). * p<0.05 Aβ_25–35/Sal <i>vs</i> PBS/Sal; # p<0.05 Aβ_25–35/Sal <i>vs</i> PBS/SP; $ p<0.05 Aβ_25–35/Sal <i>vs</i> Aβ_25–35/SP. PBS/Sal, n = 10; PBS/SP, n = 10; Aβ_25–35/Sal n = 12; Aβ_25–35/SP, n = 10.</p