Fig 1.
PS48 or Insulin restores Akt activation in β-amyloid expressing cells.
1A, left. SH-SY5Y cultures were infected with Adenovirus (Adv) encoding Aβ42 (24–36 hrs) and induced with Doxycycline (46 additional hrs). Amyloid bearing cells show inhibited Akt phosphorylation (lane 2 arrow). PS48 (1 μM) or high dose Insulin (100 nM) added 2 hrs before doxycycline induction (pre-treatment) restores Akt activation levels in cells expressing Aβ42 (lane 4 and 6). Low dose Insulin 40 nM (stimulation) is added to all cultures 20 min prior to harvest. PS48 alone does not over-stimulate Akt (lane 3 vs. 1). 1A, right. Quantification of restored inhibited Akt activation by PS48 and Insulin. pT308 Akt levels are normalized RFU, (relative fluorescence units). n = 3 experiments. Error bars are 1 SE relative to lane 1 (control). Brackets indicate individual t-tests comparing Aβ expression result (lane/bar2) to: control (lane/bar1), PS48 (lane/bar 4) and Insulin (lane/bar 6) additions. *** p < .001, **p = .01,* p < .05, t-test. Insulin increases pAkt T308 over control (p = .02, lane/bar 5 vs.1). PS48 also has a stimulatory effect (p < .01, lane/bar 3 vs.1). ANOVA 1-way: P = .03, F3.7; 2-way: P = .06, F = 3.1(between groups), P = .49 (within treatments, ie. replicates). 1A,below, in vivo-in vitro coupled assay of Akt activity. Akt was immunoprecipitated from SH-SY5Y cells infected with Adv. Phosphorylation of GSK3α/β consensus peptide proceeded in vitro after adding 200 μM ATP to the immunoprecipitate. PS48 pre-treatment (50 nM), added to cells 2 hrs. before Doxycycline addition, prevented the inhibtion of peptide phosphorylation by Aβ42. (Western not shown, n = 3, *p< .05 vs. control (Adv. alone). PS48 structure, left. 1B. Non-amyloid-based insulin resistance model. Primary rat cortical neurons (PCN) exposed to fatty acid neurotoxin, palmitate (300 μM). PS48 (100 nM, 24 hrs) partly reversed inhibition of Akt Ser473 phosphorylation by palmitate, in insulin-stimulated cells (20nM, 15 mins before lysis). Identical result in ceramde (50 μM)-treated PCNs, not shown). (*p < .05, n = 3). 1C. SH-SY5Y and C2C12 cells (representative Western) were used to test dose dependency of PS48 (1 μM, 100 nM, 10 nM). Aβ42 expression inhibits the insulin-stimulated phosphorylation (activation) of Akt (lanes 4 vs.3 and 6 vs.5 were combined to correspond to bar 2 vs.1). PS48-, doxycycline-, Insulin- additions and harvest as above. Data from both cell types were combined for the bar graph. PS48 100 nM and 1 μM (10 μM, not shown) each significantly overcome the Aβ42 effect (lanes 8 and 10 vs. 4 or 6). Bar graph: *** p < .005, Aβ vs Con; ++ p < .01, PS48/Aβ (bars 4 and 6) vs. vehicle/Aβ42 (bar 2). DMSO is vehicle control. (n = 3).
Fig 2.
PS48 or Insulin partly restores partial viability in β-amyloid expressing cells.
2A. SH-SY5Y cultures infected with Adenovirus encoding Aβ42 and induced with Doxycycline. Amyloid bearing cells are less viable in an MTT reduction assay (bar 2 vs. 1; t-test, p < .01). Pretreatment with insulin or PS48 partially reverses cell death to control viability levels (bars 4 and 6 compared to Con). Insulin or PS48 alone are also neurotrophic (bars 3 and 5 compared to Con, * p <05, n = 3). ANOVA 1-way: P = .003; 2-way P < .0001 (column effect). See text for treatment effect sizes (given as mean differences). 2B. In C2C12 myotubes, 48 hrs pretreatment with PS48 (10 μM) is comparable to pioglitazone (5 μM). +p < .05 vs. bar 2. 2C. Using a WST viability assay, cultured N2a cells are unaffected by PS48 at doses up to 100 μM, estimated LD50 is 250 μM. 24 hrs. exposure, n = 4.
Fig 3.
PS48 partly restores the suppression of LTP by Aβ42.
A. Day 14 rat prefrontal cortex (PFC) exposed to Aβ oligomers (0.5 μM) or Aβ plus PS48 (10 μM). Superimposed representative single recordings made under conditions 1 through 5, as noted. Control is DMSO-vehicle. EPSPs recorded from layer 2; stimuli (32 μA = ½ max ampl., 2 Hz) applied to layer 5. Aβ completely suppressed LTP (5 vs. 1). PS48 in the prsence of Aβ restored 68 ± 18% of control potentiated EPSP (1 hour perfusion, condition 2). A 3 hour application however showed near complete run-down (3). Aβ and Aβ/PS48 experiments were restored to control LTP levels after washout (4). B. Quantification of LTP data. Bar graph (left). LTP induction shown as mean % change (increase or decrease) from baseline (± SEM). n independent replicates are noted. Scatter graph (right) gives means of individual experiments and group averages as unadjusted % baseline values (normalized at 100%). All concentrations Aβ> 1 nM (high dose, 2.5–500 nM) completely abrogated LTP. PS47 is an inactive isomer control (n = 2). n replicates as shown. *** p < .0001 Aβ alone (high dose) vs control, ** p < .01 PS48 treatment vs Aβ alone (high doses), * p < .05 PS48 treatment vs. P47 control (Aβ high doses); ANOVA and Tukey’s tests (see text for additional statistical details).
Fig 4.
PS48 partly restores inhibited Akt phosphorylation (Akt activation) and GSK3β phosphorylation (Akt activity).
In vitro additions of recombinant PDK (5 ng; 4A.) or pre-immunoprecipitated PDK-1 (4B.). 4A. Aβ peptide addition near completely results in Akt dephosphorylation (lane 3). PS48 added after Aβ peptide (post) in the reaction mixture completely reverses the loss of Akt phosphorylation (lane 5 vs. 3). When pre-added (pre), PS48 partially restores pAkt levels (lane 4). Note commercial recombinant Akt lots come variably phosphorylated (lane 1), but lack enzymatic activity in control reactions until PDK is added (see Figs 4B and 6B and text) and also undergo further phosphorylation. 4B. Restoration of Akt-catalyzed phosphorylation of GSK3β peptide (lanes 5 and 6 vs. 4). 4C. Quantified trend to reverse inhibited Akt activation and activity by PS48 in the presence of 10 μM Aβ42 oligomers (ADDLs). PS48 is active in the 10 nM range. Data from pAktT308, pAktS473 and pGSK3α/β S9 experiments were normalized and pooled (n = 3–6 ea.). ’Fraction of control Akt activation’ is the [Aβ (± PS48 presence) /Control] densitometry signals ratio, where 1.0 represents full restoration.* p < .02, t-test, Aβ 10μM/0μM PS48 (n = 6) vs. combined 100 nM and ≥ 1 μM (1, 10 and 100 μM) data (n = 8). The mean difference is 0.23 ± 0.08, 95% CI = [0.41 to 0.05].
Fig 5.
Equilibrium enzymatic and binding studies.
PDK-1 and Akt-1 are Aβ42 targets. 5A. Aβ42 monomers and ADDL-oligomer preparations, at concentrations shown, exhibit saturation effects to inhibit the activation of Akt by PDK-1. In vitro assay data are presented as % inhibition of either activation of Akt (phospho-T308) by PDK-1 or of its subsequent activity to phosphorylate a GSK3β consensus peptide fused to paramyosin (’crosstide’). Densitometry results from both western blots were combined. Imax ~ 52% for both Aβ preparations: ADDL, K0.5 = 0.08 μM; monomers, K0.5 = 0.31 μM. n = 2–7 experiments each point, ± 1 SEM. 5B. In vitro radioassay (see methods). PS48 from 10 nM to 0.5 μM, gradually diminishes the inhibitory effect of Aβ (5 μM ADDL alone, bar 2, *p < .05 vs. vehicle) on the phosphorylation of GSK peptide (measured in cpm). The trend is toward control activity, not significant from bar 1 or other vehicle controls. 5C. Quantification of Aβ42 binding to kinase targets by fluorescence polarization (FP). The probe was FAM-tagged-Aβ42. Recombinant kinase titrations shown along bottom. The spectrophotometric polarization signal increases as the probe becomes more restricted by receptor binding. Bmax: Akt 0.32; PDK 0.16. IC50: Akt ~2 μM, PDK ~1 μM. 5D. Direct binding of recombinant PDK-1 and Akt-1 to tagged Aβ42 in solution is confirmed and exhibits saturation characteristics. Aβ42 was immunoprecipitated (6E10), fractionated by Western and co-precipitates are detected using anti- Akt and -PDK. Aβ42 concentration was fixed at 200 nM and detected using polyclonal R1282. Lane 1 is Aβ peptide control, lane 2 is beads without 6E10 control.
Fig 6.
In vitro screen of focused compound library based on PS48 scaffold.
6A. Compounds numbered according to SAR. Left, first generation; Right, second generation compounds. Two indices of effectiveness are tabeled: ’Fraction of control activity’ represents the observed amount (or fraction) of activation of Akt by PDK in the presence of compound with amyloid, relative to the no drug, no amyloid control (full activity = 1.00, Aβ42 alone is 0.62). ’Fraction of recovery’ is an index measure of the ability of a compound to restore the activation of Akt by PDK in the presence of Aβ, relative to the level of inhibition from Aβalone (drug-Aβ)/(control-Aβ) signals ratio. (full activity = 1.00, Aβ42 alone is 0.0). * p< 0.01 for both measures, n = 5 independent experiments each compound. PS47 is the inactive stereoisomer of PS48, bottom right. Final concentrations of both Aβ (ADDLs) and drug were 10 μM. Results are combined phosphorylations of Akt (T308, Akt activation) and pGSK3β consensus peptide substrate (S21/9, Akt enzymatic activity). 6B. Selected Westerns of in vitro reaction mixture by-products, highlighting significant GSK peptide (S21/9) and Akt (T308) phosphorylations in the presence of Aβ by compounds 508-1-25 and -31 (1st generation) and 508-1-68 (2nd generation). These ’hits’ restored activations at or better than PS48. 6C. In vivo verfication studies in SH-SY5Y cells. Addition of Doxycycline results in intracellular amyloid accumulation (6E10 blots). Compounds 25 and 68 (1 μM) intrinsically boost Akt phosphorylation to similar degrees, that remain sustained under Aβ pressure (as the case with PS48). Phospho-Akt levels, both T308 (as exampled) and S473 (not shown) were quantified and pooled for the bar graph, featuring 508-1-25. All cultures were stimulated with insulin (40 nM, 240 ng/ml) in the last 20 minutes of the Aβ- expression period. n = 3 experiments. * p < .05 vs. Con, + p < .01 vs. Aβ42.