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Generation of a human induced pluripotent stem cell–based model for tauopathies combining three microtubule‐associated protein TAU mutations which displays several phenotypes linked to neurodegeneration

García‐León, Juan Antonio, Cabrera‐Socorro, Alfredo, Eggermont, Kristel, Swijsen, Ann, Terryn, Joke, Fazal, Raheem, Nami, FatemehArefeh, Ordovás, Laura, Quiles, Ana, Lluis, Frederic, Serneels, Lutgarde, Wierda, Keimpe, Sierksma, Annerieke, Kreir, Mohamed, Pestana, Francisco, Van Damme, Philip, De Strooper, Bart, Thorrez, Lieven, Ebneth, Andreas, Verfaillie, Catherine M.·2018·Alzheimer's & Dementia
DOI 10.1016/j.jalz.2018.05.007
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Abstract

Introduction: Tauopathies are neurodegenerative diseases characterized by TAU protein–related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. Methods: To create a new in vitro tauopathy model, we generated a footprint‐free triple MAPT‐mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats‐FokI and piggyBac transposase technology. Results: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU‐mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. Discussion: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU‐mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening.

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Fig. 1 Design and generation of the triple TAU-mutant hiPSC lines. (A) General overview of the experimental design and strategy used to generate the template DNA including the double selection cassette and the introduction of the selected mutations into the genome through direct genome targeting mediated by tailored CRISPR-FokI nucleases and homologous recombination (HR) between highly similar sequences. (B) Layout of the nucleofection (NF) with CRISPR-FokI nucleases plus template DNA and HYG selection to generate iPSC clones harboring the GFP 1 selection cassette. (C) Example of junction assay (JA) PCRs to discriminate the presence or absence of the selection cassette in the MAPT gene in different clones. (D) Experimental design for excision of selection cassette by NF of the piggyBac transposase followed by FIAU negative selection. (E) JA PCRs demonstrating correct integration of the selection cassette before and after the excision of the piggyBac transposase; and random integration (RI) PCRs demonstrating absence of random cassette integration in the genome. (F) Sequencing of the MAPT gene following genetic modification demonstrating presence of the three mutations in heterozygosity. (G) MAPT -mutant hiPSCs express typical pluripotency markers as SOX2, SEEA4, NANOG, TRA1-81, OCT4, and TRA1-60. Hoechst 33258 (blue) was used as nuclear marker. Scale bars: 50 m m. (H) Results of embryoid body formation and characterization using the score card assay (three germ layer differentiation and loss of pluripotency marker gene expression). Abbreviations: bef., before; EB, embryoid body; lad., ladder; Tem., template.
Fig. 1 Design and generation of the triple TAU-mutant hiPSC lines. (A) General overview of the experimental design and strategy used to generate the template DNA including the double selection cassette and the introduction of the selected mutations into the genome through direct genome targeting mediated by tailored CRISPR-FokI nucleases and homologous recombination (HR) between highly similar sequences. (B) Layout of the nucleofection (NF) with CRISPR-FokI nucleases plus template DNA and HYG selection to generate iPSC clones harboring the GFP 1 selection cassette. (C) Example of junction assay (JA) PCRs to discriminate the presence or absence of the selection cassette in the MAPT gene in different clones. (D) Experimental design for excision of selection cassette by NF of the piggyBac transposase followed by FIAU negative selection. (E) JA PCRs demonstrating correct integration of the selection cassette before and after the excision of the piggyBac transposase; and random integration (RI) PCRs demonstrating absence of random cassette integration in the genome. (F) Sequencing of the MAPT gene following genetic modification demonstrating presence of the three mutations in heterozygosity. (G) MAPT -mutant hiPSCs express typical pluripotency markers as SOX2, SEEA4, NANOG, TRA1-81, OCT4, and TRA1-60. Hoechst 33258 (blue) was used as nuclear marker. Scale bars: 50 m m. (H) Results of embryoid body formation and characterization using the score card assay (three germ layer differentiation and loss of pluripotency marker gene expression). Abbreviations: bef., before; EB, embryoid body; lad., ladder; Tem., template.CC-BY-NC-ND · From the PDF
Fig. 2 Increased 4R TAU expression and TAU aggregation in the triple-mutant TAU cortical neurons. (A-F) qRT-PCR analysis showing expression of 3R and 4R TAU isoforms during the neuronal differentiation of both WT and triple TAU-mutant cells. (A) Relative expression to glyceraldehyde 3-phosphate
Fig. 2 Increased 4R TAU expression and TAU aggregation in the triple-mutant TAU cortical neurons. (A-F) qRT-PCR analysis showing expression of 3R and 4R TAU isoforms during the neuronal differentiation of both WT and triple TAU-mutant cells. (A) Relative expression to glyceraldehyde 3-phosphateCC-BY-NC-ND · From the PDF
Fig. 3 Altered electrophysiological activity of TAU-mutant iPSC-derived cortical neurons. (A) Representative recording (WT neuron on DIV70) of spontaneous action potentials. No current injection was applied. (B) Experimental current pulse step protocol (top panel) and representative traces (triple TAUmutant neuron at . DIV90) of membrane potential responses to current injections (lower panel). The membrane potential was clamped at approximately
Fig. 3 Altered electrophysiological activity of TAU-mutant iPSC-derived cortical neurons. (A) Representative recording (WT neuron on DIV70) of spontaneous action potentials. No current injection was applied. (B) Experimental current pulse step protocol (top panel) and representative traces (triple TAUmutant neuron at . DIV90) of membrane potential responses to current injections (lower panel). The membrane potential was clamped at approximatelyCC-BY-NC-ND · From the PDF
Fig. 4 Altered cortical neuron differentiation from TAU-mutant iPSCs. (A-G) qRT-PCR analysis for deep and superficial cortical layer neuron marker gene expression during differentiation of WT (black) and TAU-mutant iPSC (gray) progeny ( B3TUB , FOXG1 , TBR1 , CTIP2 , BRN2 , CUX1 , and SATB2 ). Gene expression levels are shown as fold changes relative to WT NPCs from DIV32 (gene expression was normalized to the housekeeping gene GAPDH ). (H) Graphical representation of different cortical layer marker genes. (I-L) Representative pictures for immunostaining of TUJ1 together with FOXG1, TBR1, CITP2, and SATB2 in WT (upper row) and TAU-mutant progeny at the specified time points (middle row). Quantification levels of each marker during differentiation for both cell types (WT in black and TAU-mutants in gray) depicted in the lower row. (M) DIV40 and DIV68 differentiating cells were incubated with EdU for 3 hours, 24 hours before fixation, followed by staining with antibodies against EdU and TUJ1. A representative picture of both WT and TAU-mutant neuronal progeny on DIV40 is shown, with quantification of the percentages of EdU 1 cells at both time points of WT (black) and TAU-mutant cells (gray). Hoechst 33258 (blue) was used as nuclear marker. Scale bar: 50 m m. Data are represented as mean 6 SEM of N 5 3-4 independent experiments. * P , .05, ** P , .01.
Fig. 4 Altered cortical neuron differentiation from TAU-mutant iPSCs. (A-G) qRT-PCR analysis for deep and superficial cortical layer neuron marker gene expression during differentiation of WT (black) and TAU-mutant iPSC (gray) progeny ( B3TUB , FOXG1 , TBR1 , CTIP2 , BRN2 , CUX1 , and SATB2 ). Gene expression levels are shown as fold changes relative to WT NPCs from DIV32 (gene expression was normalized to the housekeeping gene GAPDH ). (H) Graphical representation of different cortical layer marker genes. (I-L) Representative pictures for immunostaining of TUJ1 together with FOXG1, TBR1, CITP2, and SATB2 in WT (upper row) and TAU-mutant progeny at the specified time points (middle row). Quantification levels of each marker during differentiation for both cell types (WT in black and TAU-mutants in gray) depicted in the lower row. (M) DIV40 and DIV68 differentiating cells were incubated with EdU for 3 hours, 24 hours before fixation, followed by staining with antibodies against EdU and TUJ1. A representative picture of both WT and TAU-mutant neuronal progeny on DIV40 is shown, with quantification of the percentages of EdU 1 cells at both time points of WT (black) and TAU-mutant cells (gray). Hoechst 33258 (blue) was used as nuclear marker. Scale bar: 50 m m. Data are represented as mean 6 SEM of N 5 3-4 independent experiments. * P , .05, ** P , .01.CC-BY-NC-ND · From the PDF
Fig. 5 Genome-wide transcriptome studies demonstrate significant differences between triple TAU-mutant and WT neurons. RNA sequencing (seq) was performed on WT and TAU-mutant iPSC progeny on DIV70 and DIV110. (A) Principal component analysis (PCA) of WT and TAU-mutant iPSC progeny on DIV70 and DIV110. (B) Volcano plots at DIV70 (left) and DIV110 (right) illustrating the number of differentially expressed genes (adjusted P value , .05
Fig. 5 Genome-wide transcriptome studies demonstrate significant differences between triple TAU-mutant and WT neurons. RNA sequencing (seq) was performed on WT and TAU-mutant iPSC progeny on DIV70 and DIV110. (A) Principal component analysis (PCA) of WT and TAU-mutant iPSC progeny on DIV70 and DIV110. (B) Volcano plots at DIV70 (left) and DIV110 (right) illustrating the number of differentially expressed genes (adjusted P value , .05CC-BY-NC-ND · From the PDF
Fig. 6 Increased stress pathway gene expression in TAU-mutant cortical progeny. (A-M) qRT-PCR analysis for genes implicated in oxidative stress (A-D), ER/ UPRstress (E-I), and inflammatory responses (WT: black and TAU-mutant iPSC: gray, J-M). Gene expression levels are shown as fold changes relative to WT NPCs on DIV32 (gene expression was normalized to the housekeeping gene GAPDH ). (N-P) Western blot experiments and quantification of protein levels for
Fig. 6 Increased stress pathway gene expression in TAU-mutant cortical progeny. (A-M) qRT-PCR analysis for genes implicated in oxidative stress (A-D), ER/ UPRstress (E-I), and inflammatory responses (WT: black and TAU-mutant iPSC: gray, J-M). Gene expression levels are shown as fold changes relative to WT NPCs on DIV32 (gene expression was normalized to the housekeeping gene GAPDH ). (N-P) Western blot experiments and quantification of protein levels forCC-BY-NC-ND · From the PDF
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CC-BY-NC-ND · From the PDF

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Metadata

Authors
García‐León, J. A., Cabrera‐Socorro, A., Eggermont, K., Swijsen, A., Terryn, J., Fazal, R., Nami, F., Ordovás, L., Quiles, A., Lluis, F., Serneels, L., Wierda, K., Sierksma, A., Kreir, M., Pestana, F., Van Damme, P., De Strooper, B., Thorrez, L., Ebneth, A., & Verfaillie, C. M.
Venue
Alzheimer's & Dementia
Publisher
Wiley
Published
2018-07-20
Volume
14
Issue
10
Pages
1261-1280
ISSN
  • 1552-5260
  • 1552-5279
DOI
10.1016/j.jalz.2018.05.007
Cited by
50
References
10
Language
en
Update notices
correction (2020-04-22)
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)

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