Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming

Abstract

Late-onset Alzheimer's disease (LOAD) is the most common form of Alzheimer's disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-β (Aβ) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)-based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aβ-dependent neurodegeneration, and treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aβ deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

Fig. 1.. Direct reprogramming of fibroblasts from individuals with AD into 3D-CNs and neuronal spheroids and ADAD 3D-CNs and spheroids showed higher amount of Aβ deposits.

(A) A schematic diagram of 3D-direct reprogramming of patient fibroblasts to cortical neurons in thin gel culture. (B) Representative images of a thin gel culture (PID28) in one whole well of a 96-well plate immunostained with TUBB3. (C) A LONGO plot depicting increased long gene expression (LGE) during neuronal reprogramming in thin gel. (D) Heatmap shows downregulation of fibroblast markers and upregulation of neuronal markers during neuronal reprogramming in thin gel. Pan-neu: Pan-neuronal markers. Syn: Synaptic markers. Two replicates from HC83 line (healthy individual) were used for analysis in C and D. (E) A schematic diagram of direct reprogramming of human fibroblasts to neuronal spheroids. (F) A representative image of a neuronal spheroid at PID28 stained with TUBB3 depicting neurite extensions radiating out from the core of the spheroids. (G) Expression of long genes in PID28 HC spheroids, and their starting fibroblasts. (H) A heatmap shows gene expression of fibroblast markers and neuronal markers in PID28 neuronal spheroids and their starting fibroblasts. Two replicates from HC83 line were used in G & H. (I) Extracellular Aβ deposits detected by 82E1 Aβ antibody in thin gel at PID30. Aβ deposits with size > 1000 μm³ were used for quantification. (J) Immunofluorescence images (left) and enlarged 3D reconstruction images (right) showing extracellular Aβ deposition (6E10) in thin gel at PID30. Aβ deposits with size > 1000 μm³ were used for quantification. (K) Aβ42 amounts were measured by electrochemiluminescence assay in ADAD and HC spheroids at PID28. (L) Whole mount immunostaining of Aβ in ADAD spheroids and HC spheroids at PID28. Aβ deposits with size > 1000 μm³ were used for quantification. (M) Representative images of spheroid sections (PID28) and sections of 5XFAD mouse brain cortex region (3-month-old) showing the extracellular Aβ deposition. For quantification in I, J, K, and L, n = 4 ADAD and 4 HC individuals, and 2–3 thin gels or spheroids per individual line were used. * p < 0.05 and ** p < 0.01 were calculated by unpaired t-test. Scale bar: 250 μm in B and F, 50 μm in I, J, and M, and 500 μm in L.

Fig. 2.. Tauopathy, neurodegeneration, and transcriptomic features in ADAD 3D-CNs and spheroids.

(A) Immunostaining of p-tau (AT8 antibody) in ADAD and HC 3D-CNs (PID30). (B) Immunofluorescence images of pathogenic tau by co-staining of K63-linked ubiquitin and p-tau (PHF1) in ADAD and HC 3D-CNs. Arrows point to the enlarged beaded neurite bulges. (C) Transmission EM images of healthy neurites (top) and dystrophic “beaded” neurites (bottom) from HC and ADAD 3D-CNs, respectively. (D) Live-cell FRET images for detecting seed-competent tau from HC and ADAD 3D-CNs at PID26. Mean ± SEM. (E) Two spheroids were labeled by RFP or GFP and co-cultured starting at PID7. At PID17 and PID33, multiple live images were taken and compiled by Photoshop to display the whole spheroids. Left: Two spheroids derived from two different HC individuals. Right: ADAD spheroids were co-cultured with HC spheroids. HC G: HC spheroids with GFP; HC R: HC spheroids with RFP. Mean ± SEM. (F) TUNEL staining in ADAD or HC spheroid sections. 4 confocal images were taken from different areas on each section for 3 sections covering different planes of the spheroid. (G) TUBB3 immunostaining images show neurite outgrowth from the core of HC and ADAD spheroids at PID22 and PID28. Yellow dashed lines indicate the border between the core and the neurites. (H). A volcano plot displaying differentially expressed genes (DEGs) between ADAD and HC spheroids at PID25. 1411 DEGs were identified (p < 0.05, |log2fold change| > 0.58, base mean > 1). 24 samples from 4 ADAD and 4 HC individuals were analyzed. (I) Top gene ontology (GO) terms associated with upregulated and downregulated DEGs analyzed by DAVID. For quantification in A, B, D, F, and G, n = 3 or 4 ADAD and 3 or 4 HC individuals, and 2–3 thin gels or spheroids per individual line were used. For E: n = 3 pairs and 2–3 co-cultures per pair were used. Unpaired t-test for A, B, D, and F, adjusted p-values by two-way ANOVA with Šídák’s multiple comparisons test for E, and multiple paired t-tests for G. For all data: ns = p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bars: 50 μm in A and F, 25 μm in B and D, 800 nm in C, 1 mm in E, and 250 μm in G.

Fig. 3.. Elevated Aβ deposition and pathogenic tau in LOAD 3D-CNs and spheroids.

(A) Aβ deposits in thin gel culture of PID30 LOAD and HC neurons. Boxed regions were highlighted on the right by 3D reconstruction showing extracellular Aβ deposition. Aβ deposits with a size > 1000 μm³ were used for quantification. (B) Whole mount immunostaining with Aβ antibody (6E10) in PID28 LOAD and HC spheroids. Aβ deposits with a size > 1000 μm³ were used for quantification. Boxed regions were magnified at the right panels to show Aβ deposition. (C) Immunostaining images of Aβ deposits on the sections of PID28 LOAD and HC spheroids using 6E10 Aβ antibody. (D) PCR analysis of 3R and 4R tau isoforms in LOAD and HC 3D-CNs at PID25. Numbers above the gel images are sample IDs. Data was shown as Mean ± SEM. (E) Immunostaining of p-tau (AT8 antibody) in LOAD and HC 3D-CNs at PID30. (F) Co-staining of K63-specific ubiquitin and p-tau (PHF1) in the neurites (PID30 3D-CNs). Arrows highlight the swelled dystrophic neurite bulges. (G) Live-cell imaging of FRET signal from HC and LOAD 3D-CNs containing Ruby2 and Clover reporters at PID28. For quantifications: n = 5– 6 LOAD and 5–6 HC individuals and 2–3 thin gels or spheroids per individual line were used in A, B, D, E, F and G. Unpaired t-tests were used for calculating p-values (* p < 0.05, ** p < 0.01 and ns p > 0.05). Scale bars: 50 μm in A and E, 500 μm in B, and 25 μm in C, F, and G.

Fig. 4.. Spontaneous neurodegeneration in LOAD 3D-CNs and spheroids.

(A) Sytox-Green staining labeling dead cells in LOAD and HC 3D-CNs at different PIDs. Solid lines show the mean of each group whereas dotted lines represent each individual. Dead cells in LOAD 3D-CNs at PID20, PID30 and PID35 were compared to the HC 3D-CNs at the same PIDs. Mean ± SEM. p-values: two-way ANOVA with Šídák’s multiple comparisons. (B) LOAD spheroids and HC spheroids were co-cultured at PID7 and fluorescence images were taken at PID17 and PID33. Spheroid size (area, μm²) at PID33 was normalized to its size at PID17. Multiple live images were taken and stitched to display the whole spheroid. Mean ± SEM. Two-way ANOVA with Šídák’s multiple comparisons test. (C) TUNEL staining on sections of LOAD and HC spheroids (PID28). 4 confocal images were taken from different area on each section for 3 sections covering different planes of the spheroid. p-values: unpaired t-test. (D) The neurite outgrowth at proximal, middle, and distal regions in LOAD and HC spheroids was examined by immunofluorescence with TUBB3 antibody. TUBB3 signals in each region of the neurites were compared to the same region in HC89 spheroid at PID22 (set as 100%). Mean ± SEM. Multiple paired t-tests were calculated. For all quantifications: n = 5–6 LOAD and 5–6 HC individuals and 2–3 thin gels or spheroids per individual line were used. ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Scale bars: 50 μm in A and C, 1 mm in B, and 250 μm in D.

Fig. 5.. Effects of inhibiting APP processing in LOAD 3D-CNs and spheroids on tauopathy and neurodegeneration.

(A) LOAD spheroids were treated with β-secretase inhibitor IV or DAPT starting at PID16 (before the observed onset of Aβ deposition) or PID22 (after the onset of Aβ deposition) and Aβ deposition was examined at PID28 by whole mount immunostaining using Aβ antibody (6E10). (B) Tau phosphorylation (AT8, top panel) and K63-ubiquitin/p-tau (PHF1) colocalization (bottom panel) were examined in PID28 LOAD 3D-CNs treated with β-secretase inhibitor IV or DAPT starting at PID16 or PID22. Arrows highlight swelled tau blebs in the bottom panel. (C) TUNEL staining of the sections of PID28 LOAD spheroids that were treated with β-secretase inhibitor IV or DAPT starting at PID16 or PID22. For quantifications in A, B, and C, n = 5 LOAD individuals and 2–3 thin gels or spheroids per line were used. Adjusted p-values were calculated by two-way ANOVA with Šídák’s multiple comparisons test. * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bars: 500 μm in A, 25 μm in B. and 50 μm in C.

Fig. 6.. LOAD spheroids display differentially expressed genes (DEGs) and differentially expressed TEs (DETEs)

(A) Volcano plot depicts differentially expressed genes between LOAD and HC spheroids. A total of 28 spheroid samples derived from 5 LOAD and 5 HC individuals were used for analysis. Each sample contains RNAs pooled from 2–3 spheroids. Down-regulated genes (p < 0.05, log2 (fold change) <−0.58), base mean > 1; Up-regulated genes (p < 0.05, log2 (fold change) > 0.58, base mean > 1). (B) Gene annotation of disease terms linked with all the significant DEGs analyzed by DisGeNET (C) Top GO terms associated with upregulated and downregulated DEGs analyzed by DAVID. (D) A heatmap of significant (p < 0.05, |log₂fold change| > 0.58) differentially expressed transposable elements (DETEs) in PID25 LOAD and HC spheroids (divided by two age groups) analyzed by RNA-seq. 270 DETEs were identified.

Fig. 7.. 3TC (lamivudine), a reverse transcriptase inhibitor, ameliorates LOAD neuropathologies

(A) Top: Schematic of 3TC treatment for inhibiting RTE formation in LOAD spheroids. Bottom: Immunofluorescent images of ssDNA detected in young HC, old HC, LOAD, and 3TC-treated LOAD spheroids. (B) TUNEL staining for cell deaths in 3TC- or H₂O- (control) treated spheroids (PID28). (C) Whole mount immunostaining images of Aβ deposition in 3TC and H₂O-treated spheroids at PID28. (D) p-Tau labeled by AT8 antibody (top panel) and co-localization of p-tau (PHF1) and K63-ubiquitin (bottom panel) in 3TC- or H₂O-treated LOAD 3D-CNs at PID28. Arrows highlight the dystrophic, bulged tau blebs. (E) Double-stranded DNA breaks labeled by 53BP1 antibody in age-matched HC, LOAD, and 3TC-treated LOAD spheroids. (F) Volcano plot of DEGs between 3TC and H₂O-treated spheroids at PID28. 3 biological replicates per cell line, 4 independent LOAD cell lines were used per treatment (3TC or H₂O), and a total 24 spheroid samples were used for the analysis. (G) Top GO terms for all DEGs (p < 0.05, |log2 (fold change)| > 0.58, base mean > 1) analyzed by DAVID. For quantifications: n = 4 HC or/and LOAD lines and 2–3 thin gels or spheroids per line were used in A, B, C, D, and E. Adjusted p-values in A and E were calculated by one-way ANOVA with Tukey multiple comparisons test. p-values in B, C, and D were calculated by paired t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Scale bar: 10 μm in A and E, 50 μm in B, 500 μm in C and 25 μm in D.