Journal: Cell

Article Title: Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

doi: 10.1016/j.cell.2024.03.015

Figure Lengend Snippet: Figure 3. Tau propagation in 4R-P301S neurons is associated with endolysosomal dysfunction (A) Representative TEM images from soma of D35 4R neurons, D40 4R-P301S neurons, and 1.5 mg/mL K18-seeded D40 (7 + 33) 4R-P301S neurons. Scale bar, 1 mm. (B) Quantification of #MLB/neuron from D35 4R and 4R-P301S, D40 4R and 4R-P301S, and D43 (7 + 36) 4R-P301S + K18 neurons (3 mg/mL) from TEM images. n = 16 from two/ three independent experiments with eight neurons each. ***p < 0.001, one-way ANOVA, Tukey post-hoc test. (C) Representative TEM images from soma of 3 mg/mL K18-seeded D43 (7 + 36) 4R-P301S neurons at different magnifications. Scale bars left to right: 2 mm, 500 nm, 200 nm. (D) Representative TEM images from neuronal processes of 3 mg/mL K18-seeded D35 (7 + 28) 4R and 4R-P301S neurons. Scale bars left to right: 500 nm, 500 nm, 200 nm, and 100 nm. (E) VAMP7 dominant-negative (VAMP7DN) functional knockdown mechanism. TM, transmembrane. (F) Strategy for VAMP7 functional knockdown and analysis of 4R-P301S neurons. (G) Quantification of total Tau secreted from 4R and 4R-P301S neurons ±3 mg/mL K18 and overexpressing GFP or GFP-VAMP7DN incubated with 50 mM KCl. n = 7–8 from two independent experiments. **p < 0.01, one-way ANOVA, Tukey’s post-hoc test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Tau (Total) ELISA Kit Thermo Fisher Scientific Cat#KHB0042 NucleoBond Xtra Maxi EF Kit Macherey-Nagel Cat#740424.5 Laminin coated coverslips Neuvitro Corporation Cat#GG-12-15-Laminin ToxinSensor Chromogenic LAL Endotoxin Assay Kit GenScript Cat#L00350C VECTASHIELD Antifade Mounting Medium without DAPI Vector Laboratories Cat#H-1000-10 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200-10 NucleoSpin Blood L kit Macherey Nagel Cat#740954.20 MOM Immunodetection Kit Vector Laboratories Cat#BMK-2202 Deposited data Raw bulk RNA-seq data This paper GEO: GSE255902 Raw single-cell RNA-seq data This paper GEO: GSE255904 Experimental models: Cell lines Human: HEK293T line ATCC Cat#CRL-11268 Human: i3N iPSC line Wang et al.15 N/A Experimental models: Organisms/strains Mouse: Human Tau P301S (PS19) transgenic mice Jackson Laboratory Cat#008169 Oligonucleotides Primers for sequencing; see Table S1 This paper N/A Primers for qPCR; see Table S6 This paper N/A Recombinant DNA pCDH-EF1-FLPe Kazuhiro Oka Addgene #72262 psPAX Didier Trono Addgene #12260 pMD2.G Didier Trono Addgene #12259 pMDLg/pRRE Didier Trono Addgene #12251 pRSV-REV Didier Trono Addgene #12253 pC13N-CLYBL-CAG-dCas9-BFP-KRAB Martin Kampmann Addgene #127968 pMK1334 Martin Kampmann Addgene #127965 pFUGW-hSyn-jGCaMP8f This paper N/A PFUGW-hSyn-hM4D(Gi)-mCherry This paper N/A pFUGW-eGFP Manu Sharma Gift pFUGW-GFP-VAMP72-120 (VAMPDN) Manu Sharma Gift Software and algorithms R 4.2.2 The R project https://www.r-project.org/ RStudio 2022.07.2 RStudio: Integrated Development for R. RStudio https://rstudio.com Gene set enrichment analysis (GSEA) Subramanian et al.70 https://www.gsea-msigdb.org/gsea/ msigdb/index.jsp ClueGO Bindea et al.71; Shannon et al.72 https://apps.cytoscape.org/apps/cluego Adobe Illustrator Illustrator v26.5.2 https://www.adobe.com/products/ illustrator.html GraphPad Prism 6 Prism v9.2 https://www.graphpad.com FlowJo FlowJo v10 https://www.flowjo.com/ Cell Ranger- 6.1.2 10x Genomics https://support.10xgenomics.com/ single-cell-gene-expression/software/ pipelines/latest/what-is-cell-ranger (Continued on next page) ll e3 Cell 187, 1–19.e1–e13, May 9, 2024 Please cite this article in press as: Parra Bravo et al., Human iPSC 4R tauopathy model uncovers modifiers of tau propagation, Cell (2024), https://doi.org/10.1016/j.cell.2024.03.015 Article

Techniques: Dominant Negative Mutation, Functional Assay, Knockdown, Incubation

Journal: Cell

Article Title: Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

doi: 10.1016/j.cell.2024.03.015

Figure Lengend Snippet: Figure 4. Tau inclusions impair spontaneous and evoked neuronal activity (A) Strategy for targeting HaloTag at the 50 end of the MAPT locus in the 4R-P301S iPSC line. (B) Representative fluorescence image of 3 mg/mL K18-seeded 4R-P301S-HaloTag neurons (D24) harboring Tau inclusions incubated with JFX549-HaloTag ligand. Scale bar, 50 mm. (C) Representative fluorescence images expressing GCaMP8f, HaloTag, and overlay in D24 K18-seeded 4R-P301S-HaloTag spontaneous activity. Scale bar, 50 mm. (D) Quantification of spike amplitude, firing rate, and synchrony index from calcium imaging of K18-seeded 4R-P301S-HaloTag neurons (D24–D30). n = inclusion/+inclusion for spike amplitude (74/121), firing rate (76/122), and network (14/16) from 3 biological replicates. *p < 0.05, unpaired t test. (E) Representative fluorescence images of GCaMP8f, HaloTag, and overlay in D30 K18-seeded 4R-P301S-HaloTag treated with 50 mM KCl. Scale bar, 50 mm. (F) Representative averaged calcium traces from one KCl stimulation experiment in 4R-P301S-HaloTag neurons (D30) with and without Tau inclusions. Recording is 400 s. n = 11 inclusion and 8 +inclusion. Mean ± SEM. (G) Quantification of peak amplitude from KCl-stimulation-induced neuronal responses. n = 33 inclusion and 20 +inclusion from 3 biological replicates. *p < 0.05, unpaired t test. (H) Strategy for sustained neuronal activity repression and analysis of 4R-P301S-HaloTag neurons. (I) Representative spontaneous calcium traces (right) of each cell from D36 4R-P301S expressing hM4Di (left) upon 10 mM CNO addition at 60 s. Scale bar, 50 mm. (J) Quantification of MC1 area/DAPI+ nuclei from immunofluorescence images of chronically silenced D29 K18-seeded 4R-P301S neurons. n = 50 CNO and 30 +CNO from 10 images; 5 CNO biological replicates and 3 +CNO biological replicates. **p < 0.01, unpaired t test. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Tau (Total) ELISA Kit Thermo Fisher Scientific Cat#KHB0042 NucleoBond Xtra Maxi EF Kit Macherey-Nagel Cat#740424.5 Laminin coated coverslips Neuvitro Corporation Cat#GG-12-15-Laminin ToxinSensor Chromogenic LAL Endotoxin Assay Kit GenScript Cat#L00350C VECTASHIELD Antifade Mounting Medium without DAPI Vector Laboratories Cat#H-1000-10 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200-10 NucleoSpin Blood L kit Macherey Nagel Cat#740954.20 MOM Immunodetection Kit Vector Laboratories Cat#BMK-2202 Deposited data Raw bulk RNA-seq data This paper GEO: GSE255902 Raw single-cell RNA-seq data This paper GEO: GSE255904 Experimental models: Cell lines Human: HEK293T line ATCC Cat#CRL-11268 Human: i3N iPSC line Wang et al.15 N/A Experimental models: Organisms/strains Mouse: Human Tau P301S (PS19) transgenic mice Jackson Laboratory Cat#008169 Oligonucleotides Primers for sequencing; see Table S1 This paper N/A Primers for qPCR; see Table S6 This paper N/A Recombinant DNA pCDH-EF1-FLPe Kazuhiro Oka Addgene #72262 psPAX Didier Trono Addgene #12260 pMD2.G Didier Trono Addgene #12259 pMDLg/pRRE Didier Trono Addgene #12251 pRSV-REV Didier Trono Addgene #12253 pC13N-CLYBL-CAG-dCas9-BFP-KRAB Martin Kampmann Addgene #127968 pMK1334 Martin Kampmann Addgene #127965 pFUGW-hSyn-jGCaMP8f This paper N/A PFUGW-hSyn-hM4D(Gi)-mCherry This paper N/A pFUGW-eGFP Manu Sharma Gift pFUGW-GFP-VAMP72-120 (VAMPDN) Manu Sharma Gift Software and algorithms R 4.2.2 The R project https://www.r-project.org/ RStudio 2022.07.2 RStudio: Integrated Development for R. RStudio https://rstudio.com Gene set enrichment analysis (GSEA) Subramanian et al.70 https://www.gsea-msigdb.org/gsea/ msigdb/index.jsp ClueGO Bindea et al.71; Shannon et al.72 https://apps.cytoscape.org/apps/cluego Adobe Illustrator Illustrator v26.5.2 https://www.adobe.com/products/ illustrator.html GraphPad Prism 6 Prism v9.2 https://www.graphpad.com FlowJo FlowJo v10 https://www.flowjo.com/ Cell Ranger- 6.1.2 10x Genomics https://support.10xgenomics.com/ single-cell-gene-expression/software/ pipelines/latest/what-is-cell-ranger (Continued on next page) ll e3 Cell 187, 1–19.e1–e13, May 9, 2024 Please cite this article in press as: Parra Bravo et al., Human iPSC 4R tauopathy model uncovers modifiers of tau propagation, Cell (2024), https://doi.org/10.1016/j.cell.2024.03.015 Article

Techniques: Activity Assay, Incubation, Expressing, Imaging

Journal: Cell

Article Title: Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

doi: 10.1016/j.cell.2024.03.015

Figure Lengend Snippet: Figure 5. Identification of Tau inclusion modifiers by CRISPRi screening (A) Strategy for CRISPRi screening based on Tau inclusions in 4R-P301S-dCas9 neurons. (B) Volcano plot summarizing knockdown phenotypes and statistical significance (Mann-Whitney U test) for genes targeted in the pooled screen. Dashed lines are cutoff for hit genes (FDR = 0.05). (C) Subcellular classification of gene hits (product above 2.5 and below 2.5; see STAR Methods). (D and E) Representative immunofluorescence images (D) and quantification (E) of the percentage of DAPI+/MC1+ cells in D21 (7 + 14) 1.5 mg/mL K18-seeded 4R- P301S;VPS29/ neurons from clone #1 and #2 compared to the parent line. n = 4 replicates from one independent experiment per clone. ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test. Scale bar, 50 mm. (F and G) Representative flow cytometry analysis (F) and quantification (G) of the percentage of MC1+ cells in D21 (7 + 14) 1.5 mg/mL K18-seeded 4R- P301S;VPS29/ neurons from clone #1 and clone #2 compared to the parent line. N = 6 replicates from one independent experiment per clone. ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test. See also Figures S5 and S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human Tau (Total) ELISA Kit Thermo Fisher Scientific Cat#KHB0042 NucleoBond Xtra Maxi EF Kit Macherey-Nagel Cat#740424.5 Laminin coated coverslips Neuvitro Corporation Cat#GG-12-15-Laminin ToxinSensor Chromogenic LAL Endotoxin Assay Kit GenScript Cat#L00350C VECTASHIELD Antifade Mounting Medium without DAPI Vector Laboratories Cat#H-1000-10 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories Cat#H-1200-10 NucleoSpin Blood L kit Macherey Nagel Cat#740954.20 MOM Immunodetection Kit Vector Laboratories Cat#BMK-2202 Deposited data Raw bulk RNA-seq data This paper GEO: GSE255902 Raw single-cell RNA-seq data This paper GEO: GSE255904 Experimental models: Cell lines Human: HEK293T line ATCC Cat#CRL-11268 Human: i3N iPSC line Wang et al.15 N/A Experimental models: Organisms/strains Mouse: Human Tau P301S (PS19) transgenic mice Jackson Laboratory Cat#008169 Oligonucleotides Primers for sequencing; see Table S1 This paper N/A Primers for qPCR; see Table S6 This paper N/A Recombinant DNA pCDH-EF1-FLPe Kazuhiro Oka Addgene #72262 psPAX Didier Trono Addgene #12260 pMD2.G Didier Trono Addgene #12259 pMDLg/pRRE Didier Trono Addgene #12251 pRSV-REV Didier Trono Addgene #12253 pC13N-CLYBL-CAG-dCas9-BFP-KRAB Martin Kampmann Addgene #127968 pMK1334 Martin Kampmann Addgene #127965 pFUGW-hSyn-jGCaMP8f This paper N/A PFUGW-hSyn-hM4D(Gi)-mCherry This paper N/A pFUGW-eGFP Manu Sharma Gift pFUGW-GFP-VAMP72-120 (VAMPDN) Manu Sharma Gift Software and algorithms R 4.2.2 The R project https://www.r-project.org/ RStudio 2022.07.2 RStudio: Integrated Development for R. RStudio https://rstudio.com Gene set enrichment analysis (GSEA) Subramanian et al.70 https://www.gsea-msigdb.org/gsea/ msigdb/index.jsp ClueGO Bindea et al.71; Shannon et al.72 https://apps.cytoscape.org/apps/cluego Adobe Illustrator Illustrator v26.5.2 https://www.adobe.com/products/ illustrator.html GraphPad Prism 6 Prism v9.2 https://www.graphpad.com FlowJo FlowJo v10 https://www.flowjo.com/ Cell Ranger- 6.1.2 10x Genomics https://support.10xgenomics.com/ single-cell-gene-expression/software/ pipelines/latest/what-is-cell-ranger (Continued on next page) ll e3 Cell 187, 1–19.e1–e13, May 9, 2024 Please cite this article in press as: Parra Bravo et al., Human iPSC 4R tauopathy model uncovers modifiers of tau propagation, Cell (2024), https://doi.org/10.1016/j.cell.2024.03.015 Article

Techniques: Knockdown, MANN-WHITNEY, Cytometry