Fig 2: Inhibition of IDE in human neurons increases Aβ deposition in a 3D matrix(A) OX1-19 iPSC-derived neurons seeded into 3D matrigel cultures were imaged for neuronal markers MAP2 and β-III tubulin, scale bar = 200 μm. (B) Neuronal membrane potential was measured in neurons using the FLIPR® Membrane Potential Assay Kit. Representative trace of membrane potential from neurons in 3D matrigel cultures. Day 60 OX1-19 neurons were cultured in a 3D Matrigel matrix for 21 days in the absence or presence of the IDE inhibitor, ML345 (10 μM) or the NEP inhibitor, phosphoramidon (10 μM). (C) Representative images of OX1-19 neurons following treatment without or with ML345 or phosphoramidon using immunofluorescence microscopy to determine the number of Aβ deposits; composite images of βIII-tubulin and Aβ are shown and Aβ staining (with antibody 4G8) is highlighted by the arrowheads, scale bar = 200 µm. (D) Representative images of OX1-19 neurons following ML345 treatment showing only the Aβ deposits stained with antibody 4G8 with (i) being the same field of view as shown in (C), scale bar = 200 µm and (ii) a larger magnification image from a separate field of view, scale bar = 100 µm. (E) The number of Aβ deposits in each image was quantified and the spread of data from images for each condition are shown by the violin plot. Data are shown with median (solid line) and quartiles (dashed line) for 36 data points for control and ML345 and 25 data points for phosphoramidon. (F) The number of Aβ deposits was then averaged for each neuronal induction for each condition and statistical analysis performed. Data shown as mean ± SEM, n = 3 neuronal inductions (three technical repeats were performed per neuronal induction). ****P<0.0001 using an ordinary one-way ANOVA test with Dunnett’s multiple comparisons test to compared with control. (G) Representative images taken for the analysis of Aβ deposition in day 60 SBAD neurons cultured in a 3D Matrigel matrix for 21 days in the absence or presence of the IDE inhibitor, ML345 (10 μM) using immunofluorescence microscopy, scale bar = 200 µm.

Fig 3: IDE degrades Aβ in iPSC-derived neurons(A) Schematic diagram of the FAM-Aβ-biotin substrate used in the fluorescence polarisation assay. Incubation of the Aβ substrate (500 nM) with (B) 25 ng recombinant NEP (rNEP) and (C) 25 ng recombinant IDE (rIDE) in the absence or presence of the general metalloprotease inhibitor 1,10-phenanthroline (1 mM), the NEP inhibitor phosphoramidon (100 μM) or the IDE inhibitors 6bK (10 μM), ML345 (10 μM) and insulin (100 μM). NEP and IDE were pre-incubated with the inhibitors for 30 min at 37°C before addition of the FAM-Aβ-biotin substrate and a further incubation for 4 h at 37°C. The cleaved substrate was separated and the fluorescence measured as described in the Experimental section. Data shown as mean ± SEM, n = 3 experimental repeats. (D) OX1-19 iPSCs were differentiated to cortical neurons and neuronal identity was confirmed at day 80 by identification of neuronal markers using immunofluorescence microscopy. Representative images demonstrate staining for the neuronal markers MAP2 and β-III tubulin (β-III). Nuclei were stained with DAPI, scale bar represents 200 µm. (E) Membrane potential was measured in neurons using the FLIPR® Membrane Potential Assay Kit. Representative traces shown for neurons at day 60 and day 80 of differentiation. Experiments were repeated in three independent cell preparations. (F) Incubation of the Aβ substrate with human OX1-19 iPSC-derived neurons (hiPSC-Ns) in the absence or presence of the indicated inhibitors. n = 3 neuronal inductions (three technical repeats were performed per neuronal induction). **P<0.005, ****P<0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test to compared with control.

Fig 4: Aβ deposits are composed of oligomeric species and their formation is blocked by a β-secretase inhibitorDay 60 OX1-19 neurons were cultured in a 3D Matrigel matrix for 21 days in the absence or presence of the IDE inhibitor, ML345. Representative images taken for the analysis of Aβ species by immunofluorescence microscopy with conformational antibodies to detect (A) pre-fibrillar Aβ oligomers with the A11 antibody, with a higher magnification image shown in (B,C) fibrillary Aβ oligomers with the OC antibody, with a higher magnification image shown in (D). (E) Representative images of day 60 OX1-19 neurons cultured in a 3D Matrigel matrix for 21 days in the presence of the IDE inhibitor, ML345, with or without the BACE1 inhibitor βIV. (F) Quantification of Aβ deposits in OX1-19 neurons following treatment with either ML345 only or with ML345 and βIV. Scale bar in A and C = 200 µm, scale bar in B, D and E = 100 µm. Data shown as mean ± SEM, n = 3 independent cell preparations from the same neuronal differentiation *P<0.05 using a Welch’s t-test.