Fig 1: CRISPR loss of function screens identify genes and pathways required for human neuronal uptake of extracellular monomeric and fibrillar tau.Genes required for uptake of tau into human iPSC-derived excitatory neurons identified by comparing FACS collected populations of CRISPR edited neurons, positive for labelled transferrin, but not tau protein, against neurons positive for both labelled proteins. Whole-genome enrichment score (−log10 positive score) is plotted against the positive enrichment ranking from the knockout screens for tau uptake derived using the MAGeCK algorithm. Applying a p value cut-off of 0.01, 214 genes were identified as required for (A) monomeric tau uptake (purple points; two biological replicates) and 228 genes were identified as required for (C) fibrillar tau uptake (orange points; three biological replicates). The ten highest-scoring genes are labelled. Heat maps showing a representative selection of significantly enriched terms annotating the genes required for (B) monomeric and (D) fibrillar tau uptake. Grayscale indicates significance level (−log10 FDR). Rows are sorted in order of significance; see Dataset EV1 for ranked genes required for tau uptake (p < 0.01). Each heatmap shows which of the enriched genes is annotated with each term (Dataset EV2). Details of p values for each enriched term are provided in Dataset EV2. (E) Scatter plot of FACS analysis of monomeric tau and transferrin uptake by iPSC neurons individually targeted for CRISPR knockout of AP2M1 or non-targeting control (GFP). (F) Validation of genes identified in primary screens as required for monomeric tau uptake. The percentage of low tau neurons (tau-/transferrin+) is expressed as fold change from the non-targeting control (GFP). Three technical replicates were performed, across two independent experiments, represented by circles and triangles. CRISPR knockout of LRP1 and a non-targeting control (GFP) was included in both experiments (Dataset EV5). Statistical significance was determined using one-way ANOVA, Dunnett’s test for multiple comparisons (all gene perturbations presented p < 0.0001, except CCDC115 p = 0.0017). (G–I) Entry of monomeric tau into neurons is mediated by LRP1, inhibited by the addition of extracellular RAP chaperone or LRP1 domain IV peptide. The number of tau-positive objects detected over 4 h by time-lapse imaging of iPSC-derived human excitatory neurons was used to assess neuronal uptake of monomeric (A), fibrillar (B) or post-mortem AD brain (C) tau-pHrodo. Object measurements are displayed over time. Twenty-four independent measurements were taken from at least three technical replicates at 45-min intervals. Statistical significance was determined using one-way ANOVA, Dunnett’s test for multiple comparisons (monomeric tau uptake with RAP chaperone *p = 0.0228; LRP1 domain IV *p = 0.0152). See also Fig. EV2.
Fig 2: Identification of genes involved in the uptake of structurally distinct forms of tau by human cortical neurons via low-pH intracellular compartments.(A, B) CRISPR gRNA log fold change (LFC) between transferrin positive and either tau positive (+) or negative (−) neurons collected from monomeric (A) and fibrillar (B) tau uptake screens analysed using the MAGeCK algorithm. Guides for the five highest-ranked genes (gene names indicated) are highlighted on the guide density plots. Genes required for uptake of monomeric (C), fibrillar (D) tau code for proteins with a significantly higher than random localisation score in particular cellular compartments in the COMPARTMENTS dataset (FDR <0.05). Significantly enriched compartments are coloured based on the strength of enrichment (log2 fold change), whereas non-significant compartments are left white. (E) Time-lapse (0- to 4-h) images showing uptake into iPSC-derived human neurons (60 days after induction) of extracellular monomeric tau conjugated to a pH-sensitive dye (inverse relationship between fluorescence and pH). Neurons and tau protein were individually pre-incubated with either 10 nM RAP chaperone, 100 nM LRP1 domain IV peptide or vehicle control (PBS) for 3 h prior to combining the tau incubations with neurons and live imaging of neuronal uptake of tau. Bright-field (grey scale in merge) and pH-sensitive fluorescent signal (pHrodo; red in merge) were captured using automated imaging on the Opera-Phenix platform (Perkin Elmer). Scale bar, 100 μm. (F) None of the forms of tau were acutely toxic to neurons over a 16 hr period, as measured by extracellular LDH activity (three wells per treatment), in the presence of 25 nM Monomeric tau, 150 nM (monomer molar equivalent) fibrillar or post-mortem tau. (G) Extracellular LDH activity was also used to determine neuronal viability in the presence of vehicle (PBS), 10 nM RAP chaperone or 100 nM LRP1 domain IV peptide (after treatment for one week; >8 wells per treatment, across two biological replicates). Error bars indicate SD. Significance was determined using one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, Dunnett’s test for multiple comparisons). (H)The T7 endonuclease assay was used to confirm CRISPR guide RNA targeting. Assays were performed on amplified genomic DNA regions containing the target site for CRISPR gRNAs to genes indicated in the presence of targeting gRNA and the non-targeting GFP control.
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