Identification of proteins interacting with Aβ by candidate approach.

<p>(a) Western blotting with primary antibodies against synaptic markers detected specific proteins in the starting material at their expected molecular weights. Only the primary antibody against Syp detected a specific protein in the fraction eluted from the Aβ affinity column. (b) Oligomerisation state of Aβ42 compared to Aβ40 revealed by western blotting. Monomeric Aβ peptides can be seen as a band at 4 kDa. Only Aβ42 displayed visible low molecular weight aggregates.</p

Representative images demonstrating presynaptic localisation of Αβ42 (green) in mature hippocampal neurons, incubated with Aβ42 for 20 min and left to recover for various time points.

<p>After 15 min recovery, Aβ42 and Syp (<b>a</b>) or Aβ42 and VAMP2 (<b>c</b>) labelling is distinct and often juxtaposed (as detailed in high magnifications of boxed areas (right), n = 15). After 2 hrs of recovery co-localisation of Aβ42 with Syp (<b>b</b>) and VAMP2 (<b>d</b>) can be observed (n = 39 for each condition). Arrows at high magnification images of boxed area (right) point at examples of Syp (b) or VAMP2 (d) positive synaptic contacts that are also positive for Αβ42. Bracket in high magnification images of boxed area (d) shows diffuse VAMP2 staining evident after the 2 hrs recovery period. After 2 hrs of recovery Aβ42 is often juxtaposed to NMDAR (<b>e</b>) (n = 18) even after depolarisation (<b>f</b>) (n = 10). Arrows in high magnification images of boxed area point at examples of Αβ42 juxtaposed to NMDAR. Scale bar = 10 µm.</p

Disruption of the Syp/VAMP2 complex.

<p>(<b>a</b>) Immunoprecipitation of VAMP2 from hippocampal extracts assayed by western blotting for VAMP2 and Syp. A band of 19 kDa corresponding to VAMP2 and a band of 38 kDa, corresponding to Syp were detected in all conditions. A reduction in immunoprecipitated Syp was induced by Aβ42 (lane 3) compared to control (scrambled Aβ, lane 4). (<b>b</b>) The intensity ratio between Syp and VAMP2 is reduced in the presence of Aβ42 (n = 3, student t-test p = 0.0026). (<b>c</b>) co-labelling for Syp and VAMP2 in untreated hippocampal neurons, in neurons treated either with Aβ42 and allowed to recover for 15 min (<b>d</b>) or with scrambled peptide (<b>f</b>) display a significant degree of co-localisation. (<b>e</b>) Aβ42 treated neurons allowed to recover for 2 hrs display stretches of VAMP2 immunoreactivity (indicated by arrows). (<b>g</b>) Quantification of the extent of co-localisation between Syp and VAMP2. Aβ42 treated neurons left to recover for 2 hrs showed decreased level of co-localisation (M = 0.85±0.09, n = 24) compared to control (M = 0.961±0.03, n = 24, student t-test ** = 0.0003), whereas neurons left to recover for 15 min (M = 0.99±0.02, n = 24) or incubated with the scrambled peptide (M = 0.99±0.01, n = 18) did not display differences compared to control. Scale bar = 10 µm.</p

Neurotrasmitter release is affected by Aβ.

<p>(<b>a</b>) Induction of CA1- LTP after HFS in control hippocampal slices (Two-way ANOVA p = 0.0006 n = 5). (<b>b</b>) Induction of CA1-LTP after HFS is blocked in hippocampal slices after a 45 min bath application of Aβ42. (<b>c</b>) fEPSPs recorded in the CA1 in response to a 50 V stimulus of the Schaffer collateral commissural pathway showed a concentration dependent effect of Aβ in fEPSPs. Only mid-range concentrations of 0.3 nM and 1 nM were able to increase the fEPSP slope (p = 0.028 and p = 0.048 respectively with student t-test, n = 5 for each concentration tested). (<b>d</b>) Treatment with Aβ42 (45 min) increases the stimulation response compared to control, untreated slices over a range of stimuli. Results are presented as the percentage of the maximum fEPSP from control recordings (n≥5 each). Inlet in shows a trace of EPSP slopes between a control slice and a slice treated with Aβ42 stimulated at 50 V. (<b>e</b>) Chelation of Aβ42 using 6E10, a specific antibody for the peptide, abrogates the enhancement at all input values (n = 5). (<b>f</b>) Scrambled Aβ did not induce increased responses compared to controls.</p

Fusion of primed vesicles at synaptic contacts marked by labelling for Syp (green) is visualised by the extent of internalisation of FM1-43FX dye (red).

<p>High magnification images of boxed areas are shown on the right. Internalised dye at synapses is near background levels in untreated (n = 20) (<b>a</b>) as well as in neurons treated with the scrambled peptide (n = 20) (<b>b</b>). Arrows in a and b point at synaptic contacts labelled with Syp. (<b>c</b>) Exposure to Aβ42 followed by 2 hrs of recovery results in a significant increase in internalised dye at synaptic contacts (n = 20). Arrows in c point at synaptic contacts labelled with Syp. Scale bar = 10 µm.</p

ELISA assay from fractionated conditioned media (CM) from 7PA2 cells and control CHO cells detected Aβ oligomers in the conditioned medium of 7PA2 cells but not of CHO control cells (a).

<p>Hippocampal slices were incubated with CM from fractions 12, 24 and 36 of control as well as 7PA2 cells. fEPSPs were recorded in response to Schaffer collateral commissural pathway stimulation at a range of stimulus from 10 V through to 60 V (b and d). Incubation with cell-derived Aβ, increased the response to stimulation at the CA1 synapse (<b>b</b>). compared to slices treated with conditioned media from the control CHO cells (<b>d</b>). The results are expressed as a percentage of the maximum fEPSP slope from control slices. Graphs showing representative results at 50 V in slices exposed to 7 PA conditioned medium (<b>c</b>) and to control CHO conditioned medium (<b>e</b>) show that cell derived Aβ increased fEPSP (student’s t-test p = 0.003 n = 5), whereas the control medium had no affect at 50 V (n = 5) (e).</p

Representative images showing the dynamics of Aβ42 internalisation by sequential immuno-labelling.

<p>Mature hippocampal neurons incubated with Aβ42 for 20 min were left to recover for 15 min, 2 hrs and 4 hrs. (<b>a</b>) Labelling for external (red) and internalised (green) Aβ42 after 15 min recovery period. Arrows at high magnification images of boxed area (right) point at examples of overlapping staining. A reduction in co-labelling was observed after 2 hrs (<b>b</b>) or 4 hrs (<b>c</b>) recovery. Arrowheads at high magnification images of boxed area (right) show examples of external and arrows of internalised Aβ42. (<b>d</b>) Quantification of the extent of co-localisation shows a high degree of co-localisation at 15 min recovery (M = 0.92±0.07, n = 6) compared to 2 hrs (M = 0.64±0.07, student t-test ** = 0.0006, n = 10) and 4 hrs (M = 0.70±0.08, student t-test * = 0.0021, n = 8) of recovery. Scale bar = 10 µm.</p