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A three-dimensional human neural cell culture model of Alzheimer’s disease

Abstract

Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles1. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2,3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology4,5. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles6,7,8,9,10,11. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

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Figure 1: Generation of hNPCs with multiple FAD mutations.
Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.
Figure 3: Elevation of amyloid-β and p-tau levels in TBS-insoluble fractions of 3D-differentiated FAD hNPCs.
Figure 4: Detection of aggregated p-tau in the enriched ReN-mAP cells.

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Acknowledgements

This work was supported by the grants from the Cure Alzheimer’s fund (D.Y.K., S.H.C. and R.E.T.) and national Institute of Health grants 5P01AG15379 (R.E.T.) and 5R37MH060009 (R.E.T.). We thank T. L. Spires, M. Polydoro and S. Wegmann for revising the manuscript, and M. L. McKee for the electron microscopy assistance. We also appreciate B. T. Hyman, O. Berezovska, J. Hardy and P. Davies for providing cDNAs and antibodies. We acknowledge Ragon Institute’s Imaging Core facility (part of the Harvard CFAR Immunology Core), Massachusetts General Hospital (MGH) Viral Vector Core (supported by NIH/NINDS P30NS04776), MGH Microscopy Core of the Center for Systems Biology for immunoelectron microscopy (partially supported by an IBDG Grant DK43351 and a BADERC Award DK57521), MGH Confocal Microscope Core and MGH Pathology Core for technical and instrument support.

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Authors and Affiliations

Authors

Contributions

D.Y.K. and R.E.T. were equally responsible for experimental design and data interpretation. S.H.C., Y.H.K. and D.Y.K. mainly contributed to writing and revising the manuscript. D.Y.K., Y.H.K., S.H.C., M.H., S.L., C.D., H.C., C.S., B.H., J.B.K., C.A. and C.Z. conducted the experiments. S.L.W. synthesized SGSM41 and S.L.W. and C.Z. characterized SGSM41. J.M., B.J.W., M.P., C.J.W. and D.M.K. contributed to data interpretation.

Corresponding authors

Correspondence to Rudolph E. Tanzi or Doo Yeon Kim.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of FACS-sorted ReN cells with FAD mutations.

a, FACS sorting of ReNcell VM human neural stem (ReN) cells that were stably transfected with polycistronic GFP and/or mCherry lentiviral vector(s). The cells were then enriched based on GFP and/or mCherry signals by FACS (red-dotted boxes, the selected ranges of cells for the experiments; mCherry-A, area intensity of mCherry signal; GFP-A, area intensity of GFP signal). b, ReN cells stably expressing GFP alone (ReN-G), APPSL-GFP (ReN-GA), APPSL-GFP-PSEN1(ΔE9)-mCherry (ReN-mGAP), mCherry alone (ReN-m), APPSL-PSEN1(ΔE9)-mCherry (ReN-mAP) or GFP-APPSL-PSEN1(ΔE9)-mCherry (HReN-mGAP). Green, GFP; red, mCherry; scale bar, 25 μm. c, The representative fluorescence microscope images of ReN cells that were differentiated by growth-factor deprivation for 3 weeks (green, GFP; red, mCherry; scale bar, 25 μm). d, Immunofluorescence of neuronal (Tuj1) and glial markers (GFAP) in 3-week differentiated control and FAD ReN cells. Scale bar, 25 µm. e, Western blot of APPSL and PSEN1(ΔE9) expression in control (ReN-G and ReN-m) and FAD ReN (ReN-GA, ReN-mGAP and HReN-mGAP) cells. APP C-terminal fragment (CTF) levels were greatly increased by 500 nM DAPT treatments for 24 h. BACE1, β-secretase 1, F.L. APP, full-length APP. f, A table summarizing the control and FAD ReN cells generated for this study.

Extended Data Figure 2 Characterization of the control and FAD ReN cells.

a, Western blot of neuronal (MAP2, Tuj1, NCAM, synapsin 1) and glial (GFAP) markers in undifferentiated and 3-week differentiated control and FAD ReN cells. b, Confocal immunofluorescence of presynaptic (VGluT1, green) and dendritic (MAP2, red (pseudo-coloured)) markers in 6-week differentiated control ReN-m cells. Top-left, top-right and bottom-left panels, ×100 magnification; bottom-right panel represents a digitally magnified image of the respective outlined region for better visualization of punctate structures. c, qPCR array analysis of neuronal and glial markers of 7-week differentiated control ReN-G cells. Gene expression levels were normalized against β-actin levels in each sample and the fold changes were calculated by setting the expression levels of each gene in undifferentiated control ReN-G cells as 1 (n = 3 for ReN-G whereas n = 5 for ReN-G in 3D differentiation). FAD ReN cells (HReN-mGAP and ReN-mAP) showed a similar pattern of increases in neuronal and glial markers (data not shown). d, Analysis of 4-repeat (4R) or 3-repeat (3R) tau isoforms in 7-week differentiated control ReN cells (ReN-G and ReN-m) and FAD ReN cells (ReN-GA, ReN-mGAP, HReN-mGAP and ReN-mAP). Complementary DNA samples prepared from undifferentiated control ReN-G (1st lane) and human adult brains (9th lane) were used as controls. e, f, Electrophysiological properties of differentiated control ReN-G cells. The currents were elicited by 10 mV voltage steps from −100 to +60 mV in external solution (e) without (left panel in f) or with 500 nM tetrodotoxin (TTX, right panel in f). TTX treatment specifically blocked voltage-gated sodium currents. ReN-G cells were differentiated for 29 days by the previously described ‘preD’ method12. g, Sodium currents are shown as subtracted currents. h, Premature ReN-G cells (<16 days) mostly showed voltage-gated potassium currents without TTX-sensitive sodium currents. i, Western blot of amyloid-β (Aβ) levels in the conditioned media collected from 6-week differentiated control (ReN-m) and FAD ReN (ReN-mAP and HReN-mGAP) cells. j, A table summarizing APOE genotypes of control (ReN-m) and FAD ReN (ReN-mAP) cells used in this study. Two APOE SNP markers, rs429358 (minor allele = C) and rs7412 (minor allele = T), were used to determine APOE ε2/3/4 genotypes.

Source data

Extended Data Figure 3 Characterization of differentiated ReN cells in 3D cultures.

a, Haematoxylin staining of a representative paraffin section (10 μm) from 9-week differentiated ReN-m cells in thick-layer 3D Matrigel. The sections were vertically cut to show the top and bottom of the 3D cultures. The pictures (10) were serially taken from top to bottom and digitally combined together. Bottom panel represents a digitally magnified image of the respective outlined region for better visualization; scale bar, 50 µm. b, The paraffin sections from control (ReN-G and ReN-m) and FAD ReN (ReN-GA and ReN-mAP (enriched)) were immunofluorescence stained with antibodies against the neuronal markers Tuj1 and MAP2. Blue, DAPI; scale bar, 25 μm. c, Immunofluorescence of thick-layer 3D-differentiated ReN-m cells stained with antibodies against additional neuronal markers tau, VGluT1 or GluR2 (glutamate receptor, ionotropic, AMPA 2); scale bar, 25 μm. d, Immunofluorescence of 3D-differentiated ReN-m cells (7 weeks) stained with antibodies against mature neuronal markers TH (tyrosine hydroxylase), NR2B (NMDA receptor 2B) or GABA(B)R2 (GABA-B-receptor 2); blue, DAPI; scale bar, 20 μm.

Extended Data Figure 4 Reconstitution of amyloid-β aggregates in 3D-cultured FAD ReN cells.

a, IHC of amyloid-β deposits in control (ReN-G) and FAD ReN (HReN-mGAP) cells differentiated in 3D Matrigel. The control and FAD ReN cells were 3D-differentiated for 6 weeks and then treated with 1 μM BACE1 inhibitor IV (β-secretase inhibitor), 500 nM DAPT (γ-secretase inhibitor), 500 nM SGSM41 (γ-secretase modulator) or DMSO for an additional 3 weeks. The cultures were then fixed and immunostained with HRP-conjugated BA27 anti-Aβ40 antibodies (brown, DAB (BA27); blue, haematoxylin; scale bars, 25 μm; arrowhead, large amyloid-β deposits). b, Enlarged images of amyloid-β deposits in control (ReN-G) and FAD ReN (ReN-mGAP, HReN-mGAP) cell pictures shown in Fig. 2d and Extended Data Fig. 4a. Scale bar, 25 µm. c, IHC of amyloid-β deposits in control (ReN-G, left panels) and FAD ReN (ReN-mGAP, middle panels; HReN-mGAP, right panels) cells differentiated in 3D thin-layer Matrigels for 9 weeks. The fixed thin-layer 3D cultures were immunostained with HRP-conjugated BA27 anti-Aβ40 antibodies (brown, DAB (BA27); scale bar, 25 mm; arrows, large amyloid-β deposits). d, Amylo-Glo staining of ReN-G 3D cultures. Green, GFP; blue, Amylo-Glo; ×10 magnification.

Extended Data Figure 5 Accumulation of p-tau in FAD ReN cells.

a, Elevated p-tau levels were significantly decreased by 1 μM DAPT (γ-secretase inhibitor) treatment in 6-week differentiated HReN-mGAP cells. The antibody against human specific mitochondrial marker (h-mito) was used to show an equal loading of the samples. b, Quantification of p-tau levels in control (DMSO)- and DAPT-treated HReN-mGAP cells (**P < 0.01; t-test; n = 3 per each sample). y axis values represent relative signal intensities (% adjusted volumes) of each p-tau band. c, p-tau IHC showed p-tau-positive cells in 3D-differentiated FAD ReN cells. Two p-tau antibodies, AT8 (pSer 199/Ser 202/Thr 205) and PHF1 (pSer 396/Ser 404), against different phosphorylation sites were used. Brown, p-tau; scale bar, 25 μm; arrows indicate cells with high levels of p-tau. d, High magnification (×100) images of p-tau-positive neurons (brown, AT8 p-tau). IHC of AT8 p-tau staining showed neurons with high levels of p-tau accumulation in soma and neurite-like structures (arrowheads). e, IHC of p-tau (AT8) staining showed the cells with p-tau accumulations in soma and neurite-like structures (arrow and arrowhead, respectively); scale bar, 25 µm. f, A digitally enlarged image of a dotted box in e. Brown, p-tau (AT8); blue, haematoxylin; scale bar, 25 μm. g, Total number of cells with high levels of p-tau in a single well of a 96-well plate was counted in control (ReN-G and ReN-m) and FAD ReN (ReN-GA, ReN-mGAP and HReN-mGAP) cells (*P < 0.05; t-test; n = 5 for control ReN cells and n = 12 for FAD ReN cells). h, BACE1 inhibitor IV (1 μM) or compound E (3.7 nM) treatments markedly decreased the number of cells with high p-tau accumulation in ReN-mAP cell populations (***P < 0.001; ANOVA followed by a post hoc Dunnett’s test; n = 3 for control ReN-m and ReN-mAP cells).

Source data

Extended Data Figure 6 FACS enrichment of ReN-mAP and ReN-m cells for higher expressions of APP and PS1.

a, FACS sorting of ReN-mAP cells with top 1–2% mCherry signal. mCherry-A, area intensity of mCherry signal. b, Representative images of the mCherry-labelled (red), enriched control ReN-m and ReN-mAP cells before or after the differentiation under growth-factor free conditions (×200 magnification); 1- and 3-week differentiated cells show increased neurite outgrowth. c, The enriched ReN-mAP cells secreted high levels of Aβ40 and Aβ42 after 9-week 3D differentiation. The secreted Aβ38/40/42 levels were measured by a multi-array Meso Scale electrochemiluminescence (Meso Scale SQ 120 system). Relative levels of amyloid-β (fold increases) were calculated by setting amyloid-β levels of the control ReN-m as 1. Amyloid-β levels were changed after treating 1 μM BACE1 inhibitor IV, 3.7 nM compound E or 500 nM SGSM41 (**P < 0.01; ***P < 0.001; ANOVA followed by a post hoc Dunnett’s test; n = 3 for the enriched ReN-m and ReN-mAP cells).

Source data

Extended Data Figure 7 Increased p-tau levels in FAD ReN cells.

a, Immunofluorescence of AT8 p-tau and Tuj1 in the enriched ReN-mAP and control ReN-m cells after 9 weeks of 3D differentiation. BACE1 inhibitor IV treatment for 3 weeks dramatically reduced AT8 p-tau staining (green, AT8 p-tau; red (pseudo-coloured), Tuj1; scale bar, 25 μm). b, Western blot of total and p-tau levels in control (enriched ReN-m) and FAD ReN (enriched ReN-mAP) cells. The cells were 3D differentiated for 9 weeks. Three weeks of BACE1 inhibitor treatments significantly decreased p-tau levels without changing total tau levels. HSP70 heat shock protein levels are shown to demonstrate equal loading of each sample.

Extended Data Figure 8 Immunoelectron microscopy analysis of sarkosyl-insoluble fraction from FAD and control ReN cells.

a, Sarkosyl-insoluble fractions prepared from 3D-differentiated ReN-mAP (enriched, 7-week differentiated) were placed on carbon grids, labelled with tau46 and anti-mouse 10 nm gold antibodies and imaged using a JEOL JEM 1011 transmission electron microscope (scale bar, 500 nm). b, Sarkosyl-insoluble fractions from 3D-differentiated control ReN-G cells (7-week differentiated). No immunogold-labelled filamentous structures were detected in these samples (scale bar, 500 nm).

Extended Data Figure 9 Treatment with 1-azakenpaullone, a GSK3β inhibitor, decreased amyloid-β-induced tau phosphorylation without changing total amyloid-β levels.

a, Immunofluorescence of p-tau and MAP2 in the enriched ReN-mAP and control ReN-m cells with or without treatment with 1-azakenpaullone, a GSK3β inhibitor. The differentiated cells were treated with 2.5 μM 1-azakenpaullone or DMSO for the last 5 days of the 3D differentiation (green, p-tau (PHF1); red (pseudo-coloured), MAP2; scale bar, 25 μm). b, Western blot of total and p-tau levels in control (enriched ReN-m) and FAD ReN (enriched ReN-mAP) cells. The cells were 3D differentiated for 4 weeks followed by additional 5-day treatments of DMSO or 2.5 μM 1-azakenpaullone. c, Analysis of Aβ40 and Aβ42 levels in the enriched ReN-mAP cells treated with either DMSO or 2.5 μM 1-azakenpaullone (1-Aza) under the same conditions.

Source data

Extended Data Table 1 The structure and the properties of SGSM41, a novel soluble γ-secretase modulator

Supplementary information

3D reconstitution of ReN-G cells after 2-week differentiation in 3D Matrigel (green, GFP; 400x magnification)

Due to the technical limitation of spinning disk confocal microscopy (Olympus), 3D reconstituted images represent only a part of the whole 3D culture. (MP4 8366 kb)

3D reconstitution of ReN-m (enriched) cells after 4-week differentiation in 3D Matrigel (red, mCherry; 400x magnification)

3D reconstitution of ReN-m (enriched) cells after 4-week differentiation in 3D Matrigel (red, mCherry; 400x magnification) (MP4 2960 kb)

3D reconstitution of 3D-differentiated ReN-m cells for 6 weeks (green, Synapsin I; red, mCherry; 1,000x magnification).

3D reconstitution of 3D-differentiated ReN-m cells for 6 weeks (green, Synapsin I; red, mCherry; 1,000x magnification). (MP4 25832 kb)

Z-section sequences of 3D6-stained Aβ aggregates in 6-week differentiated ReN-mGAP cells (green, GFP; 400x magnification).

Z-section sequences of 3D6-stained Aβ aggregates in 6-week differentiated ReN-mGAP cells (green, GFP; 400x magnification). (MP4 3847 kb)

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Choi, S., Kim, Y., Hebisch, M. et al. A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature 515, 274–278 (2014). https://doi.org/10.1038/nature13800

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