Shifting of amidated RXFP3 agonist concentration response curves by 135PAM1 in cells lacking chimeric G proteins.

<p>SK-N-MC cells coexpressing RXFP3 and a reporter construct linking CRE activity to β-galactosidase were incubated with fixed concentrations of 135PAM1 (0, 0.2, 2 and 20 µM) and increasing concentrations of Relaxin-3_NH2 (A) or R3I5_NH2 (B).</p

135PAM1 increases the intracellular Ca²⁺ response to amidated, but not free acid RXFP3 agonists in cells coexpressing RXFP3 and G_qI5.

<p>Intracellular Ca²⁺ responses by HEK-293 cells coexpressing RXFP3 and G_qI5 were measured in response to escalating concentrations of 135PAM1 using probe (EC₂₀) concentrations of Relaxin-3_NH2 (A), Relaxin-3_OH (B), R3/I5_NH2 (C), or R3/I5_OH (D).</p

Structure of 135PAM1 (3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea).

<p>Structure of 135PAM1 (3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea).</p

135PAM1 shifts the concentration response curves of Relaxin-3_NH2 and R3/I5_NH2.

<p>HEK-293 cells coexpressing RXFP3 and G_qI5 were incubated with fixed concentrations of 135PAM1 (0, 0.2, 2 and 20 µM) 10 min before the addition of increasing concentrations of Relaxin-3_NH2 (A), R3I5_NH2 (B), Relaxin-3_OH (C) or R3I5_OH (D).</p

135PAM1 lacks affinity at the orthosteric binding site of RXFP3 receptor.

<p>135PAM1 did not displace [125I] R3/I5_NH2 at concentrations of up to 20 µM, but instead increased total binding. R3/I5_NH2 displaced the tracer with a pIC₅₀ of 8.76 (8.91 to 8.61).</p

Using Human iPSC-Derived Neurons to Model TAU Aggregation

<div><p>Alzheimer’s disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.</p></div

K18 seeding induces TAU aggregation and hyperphosphorylation in human TAU-P301L neurons.

<p>Optimal timelines are shown for AAV transduction, 96w final plating, K18 seeding and final assay. <b>(A, B)</b> AlphaLISA data show that K18 seeding induces an increase in both hTAU10 (P<0,001; A) and AT8 (P<0,001; B) TAU aggregation assays. <b>(C, D)</b> AlphaLISA results demonstrate around 2-fold increase in TAU phosphorylation (AT8/hTAU10; P<0,001; C) while total TAU levels remain unchanged (HT7/hTAU10; P = NS; D). <b>(E)</b> CellTiter-Glo® results showing that general cell health is unaffected after K18 addition (P = NS). For all assays in <b>(A-E)</b>: *** P<0,001; 1-way ANOVA with Tukey’s post hoc; n≥3 independent experiments. <b>(F)</b> Representative blot of Native PAGE followed by Western blot showing two monomeric HT7-positive TAU bands (around 66kDa) in all conditions. Notably, non-migrated HT7-positive TAU proteins (>1236kDa) in K18-seeded samples suggest the presence of TAU aggregates. <b>(G, H)</b> Representative Western blots after Sarkosyl extraction showing soluble (S) and insoluble (IS) fractions after blotting with antibodies against total TAU (HT7; G) and hyperphosphorylated TAU (AT8; H). Aggregates are only present in the insoluble pellet after addition of 6nM or 50nM of K18 fibrils. Note the presence of monomeric 3R and 4R TAU protein in the soluble fraction. <b>(I)</b> Immunostaining for AT8 and neuronal HuC/D after 1%Triton/PFA fixation, to remove monomeric TAU, reveals AT8-positive neurons after K18 seeding. Scale bar = 25μm.</p

AlphaLISA optimizations on human brain extracts for total TAU, TAU aggregation and phosphorylation.

<p><b>(A, B)</b> AlphaLISA on 2 different AD brain extracts show high hTAU10 (A) and AT8 (B) TAU aggregation signals compared to control brain samples. <b>(C, D)</b> AT8/hTAU10 (C) AlphaLISA on these AD brain extracts reveals high levels of phosphorylated TAU compared to control brain samples while both AD and control brain extracts display high HT7/hTAU10 (D) levels. Decreasing signals with increasing dilutions suggest no hooking of the samples. Representative curve of 1 experiment with 2 technical replicates is shown as RFU (relative fluorescence units) ± SD. <b>(E, F)</b> Western Blot on soluble (S) and insoluble (IS) fractions of control and AD brain extracts after Sarkosyl extraction shows HT7-positive (E) and AT8-positive (F) bands only in the Sarkosyl insoluble pellets of both AD patients, confirming the presence of TAU aggregates. M represents Magic Marker (band sizes) and T represents TAU ladder with all 6 TAU isoforms. All experiments have been confirmed at least twice.</p

Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons.

<p><b>(A)</b> CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). <b>(B, C)</b> Rapamycin dose-dependently reduces hTAU10 (P<0,001; B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM; C) and compared to DMSO. <b>(D, E)</b> Also general TAU phosphorylation is reduced (AT8/hTAU10; P<0,001; D) to a similar extent as the reduction in total TAU (HT7/hTAU10; P<0,001; E). <b>(F)</b> CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). <b>(G, H)</b> AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels <i>versus</i> control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). <b>(I, J)</b> Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. ***P<0,001; **P<0,01; *P<0,05; ^#P<0,001 due to toxicity; 1-way ANOVA with Dunnett’s post hoc; n≥3 independent experiments</p

Optimization of dynamic range of hTAU10 aggregation AlphaLISA.

<p><b>(A)</b> K18 sonication significantly improves seeding potency (P<0.001, n≥3 independent experiments). Seeds were added at week 1. In all further experiments, sonicated K18 is used. <b>(B)</b> K18 seeding does not induce aggregation in control (no virus) and WT virus (AAV WT TAU 2N4R) transduced neurons (P = NS; n≥3 independent experiments). <b>(C)</b> Weekly repeated K18 seeding of K18 significantly increases the dynamic range (P<0.001 for both 1xF <i>versus</i> 2xF (wk 1+2) and 1xF <i>versus</i> 3xF (wk1+wk2+wk3); n≥3 independent experiments). <b>(D)</b> Finally, also the timing of seeding has an effect on the aggregation potency. Addition of K18 at week 2 (wk2) significantly increases TAU aggregation compared to week 1 (P<0.001, n≥3 independent experiments) while addition of fibrils at week 3 shows significantly less aggregation (P<0.001; n≥3 independent experiments) probably due to the shorter (1 week) K18 incubation period before AlphaLISA. ***P<0,001; 2-way-ANOVA with Dunnett’s post hoc.</p