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

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 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

Chemical structures of compounds.

<p>Chemical structures of compounds.</p

Correlation of brain concentration of compounds with behavioral efficacy and estimated receptor occupancy at sigma-2/PGRMC1 receptor.

<p>CT0093 and CT0109 were dosed subcutaneously in mice by continuous osmotic minipumps infusions at the doses indicated. CT01344 and CT01346 were dosed by once daily oral gavage. Twenty four hours after the last dose, animals were euthanized and drug concentration in the brain was measured. Ki  =  binding affinity of compound at the sigma-2/PGRMC1 receptor. Measured efficacy: statistically significant improvement (+), or no significant improvement (-) seen in behavioral tests. Estimated % receptor occupancy was calculated according to the formula (concentration/Ki)/[(concentration/Ki) + 1)], where Ki is determine by radioligand competition binding.</p><p>Correlation of brain concentration of compounds with behavioral efficacy and estimated receptor occupancy at sigma-2/PGRMC1 receptor.</p

Characterization of human Abeta 1–42 oligomers isolated from patient frozen, unfixed 1 gram brain samples by non-denaturing Western blot, MALDI-TOF and ELISA.

<p><b>A</b>, Non-denaturing Western blots of immunoprecipitated Alzheimer's patient hippocampal samples demonstrates heterogeneous populations of oligomer assemblies. 6E10 antibody labeling of western blots from four different AD patients (lanes 1–4) detects major bands ≥250 kDa, and multiple discrete bands between 50–75 kDa. In contrast, APP antibody detects a faint band at 125 kDa (lane 5). Significant amounts of monomeric Abeta 1–42 were not observed in any individual. MALDI-TOF analysis of immune-precipitated human brain samples demonstrates heterogeneous populations of oligomer assemblies, both between individual Alzheimer's patients (<b>B, D</b>) and between age-matched histologically normal individuals (<b>C, E</b>). Significant amounts of monomeric Abeta 1–42 were not observed in any individual. Albumin was added to samples as an internal size control (arrow in <b>B–E</b>).</p

Small molecule Abeta binding antagonists prevent Abeta 1–42 oligomer-induced synaptic regression in cultured neurons.

<p><b>A</b>, Abeta oligomers bound to a subset of neurites (red) reduces synaptophysin-immunoreactive synaptic puncta (green). <b>B</b>, Treatment with sigma-2/PGRMC1 antagonists reduces oligomer binding and restores normal immunoreactivity for the synaptic marker. <b>C</b>, Oligomers induce an average 18%±2 s.e.m. loss in the number of immunoreactive puncta per micron length of neurite (red bar) compared to vehicle-treated cultures (blue bar). Treatment of cultures with sigma-2/PGRMC1 antagonists (closed bars) restores synaptophysin immunoreactivity to normal, but has no effect when antagonists are dosed alone (open bars). *p = 0.05, Student's paired t-test.</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