The amount of background signal with APP-KO tissue varies widely between different ELISAs.

<p><b>A</b>) The Covance Colorimetric BetaMark™ Beta-Amyloid x-40 ELISA Kit (left) and x-42 Kit (right). Both kits give a signal with APP-KO tissue that is significantly above baseline. The x-40 kit gives a signal with APP-KO tissue that is not significantly different from WT (the difference between APP-KO and WT is significant for the x-42 kit). <b>B</b>) The Invitrogen Aβ 40 Mouse ELISA Kit (left) and Aβ 42 Mouse ELISA Kit (right). Both kits give a signal with APP-KO tissue that is significantly above baseline, but also significantly different from WT tissue. <b>C</b>) The Wako β-Amyloid (40) ELISA Kit (left) and β-Amyloid (42) ELISA High-Sensitive Kit (right). Both kits give a signal with APP-KO tissue that is not statistically significant from baseline. <b>D</b>) Both WT and APP-KO give a similar level of background signal with an ELISA for human β-amyloid (6E10 capture antibody – left), but show a clear difference when a rodent-specific capture antibody is used (M3.2 antibody – right). <b>E</b>) “Signal to noise” ratio of the WT signal divided by the APP-KO signal for each ELISA. Error bars in A–D are standard error.</p

Several β-amyloid antibodies show high non-specificity for β-amyloid by western blot.

<p><b>A</b>) Western blot with the antibody M3.2 (specific for rodent β-amyloid) using hippocampal tissue from APP-KO, WT, and APP transgenic mice expressing human APP (Hu<i>APP</i>695SWE). All three genotypes show a similar band pattern. <b>B</b>) Western blot with the antibody 6E10 (specific for human β-amyloid). All three genotypes show a similar band pattern. However, with 6E10, the APP transgenic mouse tissue shows an additional 8 kD band (presumably a human β-amyloid dimer) as well as a band near 87 kD (presumably full-length human APP). In addition, a 4.5 kDa band (β-amyloid) is detected when older mouse brain is used that has high levels of β-amyloid protein. <b>C</b>) Western blot with 4G8. All three genotypes also showed a similar band pattern with this antibody as well. In addition, a 4.5 kDa band (β-amyloid) is detected when older mouse brain is used that has high levels of β-amyloid protein.</p

Effect of Abeta control peptides in membrane trafficking assay.

<p>Oligomer preparation of Abeta 1–40 is 200 times less potent than Abeta 1–42 in binding assay (<b>A</b>) and in membrane trafficking (<b>B</b>). Scrambled Abeta 1–42 is not active in the membrane trafficking assay and is not detectable by the antibody used for the Abeta binding assay.</p

Chemical structures of compounds.

<p>Chemical structures of compounds.</p

Small molecule Abeta binding antagonists do not act directly on Abeta oligomers.

<p>An ELISA specific for oligomeric forms of Abeta 1–42 shows that (A) preformed oligomers are dissociated by 8-OH quinoline but not by CT0109 or CT0093, and; (B) assembly of oligomers are inhibited by Tween but not by CT0109 or CT0093 at concentration of up to 20 µM for 24 hr, ruling out a direct effect of these compounds on oligomer assembly or disruption.</p

Characterization of synthetic human Abeta 1–42 oligomers by non-denaturing Western blot, MALDI-TOF.

<p><b>A</b>, Freshly prepared solutions of synthetic human Abeta 1–42 (lane 1) or 1–40 (lane 3) peptide loaded onto non-denaturing western gels immediately after reconstitution contain large amounts of monomer (arrow; fainter lower molecular weight band represents peptide degradation product) and little higher molecular weight material. In contrast, the same solution of Abeta 1–42 peptide that is allowed to oligomerize for 24 hours (lane 2) contains much larger amounts of higher molecular weight material >50 kDa, and less monomeric protein. The full length of gel lanes are shown from loading well to dye front. Note that oligomers run differently on non-denaturing gels than globular molecular weight protein size standards <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111898#pone.0111898-Tseng1" target="_blank">[49]</a>. <b>B</b>. The presence of significant amounts of monomer in oligomer preparations is also confirmed by MALDI-TOF analysis of the same Abeta 1–42 oligomer preparation that shows both a 4.5 kDa monomer peak and multiple lower abundance peaks corresponding to oligomers of various sizes. MALDI-TOF (detection range 3–100 kDa) of vehicle (media without Abeta) is shown below for comparison (<b>C</b>).</p

Potency of compounds in membrane trafficking assay.

<p>Prevention: compound added 1 hr prior to Abeta oligomers. Treatment: compound added 1 hr after Abeta oligomers. N =  number of experimental repeats (four replicate wells per experiment). n.d.  =  not determined.</p><p>Potency of compounds in membrane trafficking assay.</p

Small molecule Abeta binding antagonists improve cognitive deficits in mice.

<p><b>A,B</b>, sigma-2/PGRMC1 antagonists prevent oligomer-induced contextual fear conditioning memory deficits in C57BL/6 male mice. <b>A</b>. No behavioral deficits are observed during fear conditioning training with any treatment. <b>B</b>. Testing 24 hours after training reveals that a single injection (2 µM) of Abeta antagonists CT0093 (solid gray bar) or CT0109 (solid black bar) via bilateral intrahippocampal injection one hour prior to oligomer injection (200 nM) prevents oligomer-induced fear memory deficits (solid red bar;CT0109: *p = 0.03, CT0093: *p = 0.05, pairwise t-test comparing Abeta vs. Abeta plus compound). Treatment with compound in the absence of Abeta oligomers does not result in fear memory deficits (open grey and black bars, N = 10–18 animals/group). Treatment with CT01202 or CT01206 (2 µM) did not prevent Abeta oligomer-induced memory deficits (solid orange and green bars, ns  =  not significant by paired t-test comparing Abeta vs. Abeta plus compound, N = 12, 9, respectively) and caused fear memory deficits in the absence of Abeta (open orange and green bars, *p = 0.05, paired t-test, vehicle, vs compound alone, N = 11, 8 respectively). <b>C</b>. Abeta oligomer antagonists rapidly improve cognitive deficits in aged transgenic mice. Eleven month old female hAPP Swe/Ldn transgenic or wild-type littermates treated for 42 days with CT01346 at 30 mg/kg/day p.o. significantly improves transgenic animal spatial memory retrieval performance in Morris water maze probe trial (**p = 0.005, paired t-test, N = 7–9 animals/group). <b>D</b>. Abeta oligomer antagonists sustain cognitive improvement in aged transgenic mice. Nine month old male hAPP Swe/Ldn transgenic mice treated for 5.5 months with vehicle or Abeta antagonists CT01344 at 10 and 30 mg/kg/day or CT01346 at 30 mg/kg/day p.o. significantly improves transgenic animal contextual fear conditioning memory deficits (*p = 0.0237,*p = 0.25, ***p = 0.0005, respectively, Mann Whitney U test, N = 13–15 animals/group).</p

Binding affinity (Kd) of Abeta preparations to cellular compartments of neurons and glia.

<p>Mean Kd ± S.E.M. Data are results of 13 replicates for oligomer binding and 4 replicates for monomer binding. NS  =  no significant binding above background.</p><p>Binding affinity (Kd) of Abeta preparations to cellular compartments of neurons and glia.</p

Relative potency of Abeta preparations in membrane trafficking assay.

<p>Synthetic human Abeta 1–42 oligomer (high concentration), freshly made monomer, synthetic oligomers (low concentration), semi-synthetic oligomers and human Alzheimer's patient derived oligomers were dosed in the membrane trafficking assay. All Abeta preparations alter membrane trafficking rates but with different EC₅₀ concentrations and different exposure times to B_max, similar to literature reports (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111898#pone-0111898-t002" target="_blank"><b>Table 2</b></a>).</p