LRP1 is a master regulator of tau uptake and spread

Abstract

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy¹. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity². This observation and complementary experimental studies^3,4 have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.

Extended Data Figure 1. Tau uptake is regulated by LRP1.

a, Gating strategy for tau uptake assay. First, cells were gated on FSC/SSC (Mean FSC-A: ~ 8,000,000/Mean SSC-A: ~800,000). Cells were then gated on FSC-H vs. Width to discriminate doublets. Dead cells were removed from analysis using propidium iodide as a stain, and positive cells were determined by gating on a negative (no tau added) population. b, Internalization controls for tau uptake assay (n=6). c, qPCR analysis of various genes with sgRNA in H4i cells. First column represents NT sgRNA control for each target (n=3). d, Uptake of phosphorylated (p2N4R) or mutated (2N4RP301L) full length tau in H4i cells (n=8). e, Western blot analysis of LRP1 in WT, NT sgRNA or LRP1 sgRNA H4i cells. All results in b-e were performed in three independent experiments and normalized to WT uptake (100%). Data is expressed as mean ± s.d. One-way ANOVA with Tukey’s method, two-sided was performed to determine significance, ****=p-value < 0.0001.

Extended Data Figure 2. LRP1 ligands influence tau uptake.

a, Uptake of 2N4R tau with RAP or lysine capped RAP competition. b, Uptake of 2N4R tau with RAP or mtRAP competition. c, Corrected Integrated Density of surface HA staining for different ectodomain cell lines (n=60). d, Co-immunoprecipitation of HA-mLRP4 with myc-2N4R (n=3). e, Uptake of various ligands at indicated concentrations (1hr). f, Western blot analysis of conditioned media from HEK293T cells mock treated or overexpressing ApoE (n=3). g, Uptake of full-length tau and transferrin in conditioned media from f (n=9). All results in a-b,e were performed in biological duplicates over three independent experiments (n=6) with representative experiments shown. Data is expressed as mean ± s.d. One-way ANOVA with Tukey’s method, two-sided was performed to determine significance, ****=p-value < 0.0001.

Extended Data Figure 3. LRP1 expression influences tau spread.

a, qPCR analysis of LRP1 expression in iPSN (n=3; p-value < 0.0001). b, qPCR analysis of LRP1 expression from mouse cortex transduced with hSyn Scramble shRNA or LRP1 shRNA (n=3; p-value = 0.0412). c, Quantification of tau spread in animals broken down by sex (Males n=4, Females n=3; Two-way ANOVA, sex = ns, p-value = 0.5335). d, Immunofluorescence of mouse primary culture transduced with AAVmRuby-hSyn-shLRP1 (green, Tau; red, mRuby; blue, Hoechst; scale bar 20μm, n=3). d, Immunohistochemistry of hTau⁺ astrocytes in PBS injected animal (green, Sox2; red, hTau; blue, Hoechst; scale bar 20μm, n=3). e, Immunohistochemistry of scramble shRNA and LRP1 shRNA animals (green, LRP1; blue; Hoechst, scale bar 20μm, n=3). Unpaired t-test, two-tailed was performed to determine statistical significance for qPCR in a,b, *=p-value ≤0.05, ****=p-value < 0.0001.

Figure 1:. Tau uptake is mediated by LRP1.

a, Diagram of tau uptake assay. Fluorescently labeled tau is added to the cellular medium where it can be endocytosed by cells. The uptake of tau can be measured via fluorescence using flow cytometry of single cells. See methods for more details. b, Flow cytometry measurement of tau uptake (50nM, 1hr) in H4 CRISPRi neuroglioma cells (H4i) with various LDLR family proteins knocked-down, NT sgRNA=Non-targeting sgRNA (n=12, LRP5/LDLR n=9). c, Tau oligomer and sonicated fibril endocytosis in H4i cells (50nM, 100nM respectively, 1hr, n=11). d, Transferrin uptake (500nM, 1hr, n=9) in H4i cells (ANOVA; p-value = 0.4464). e, Tau isoform endocytosis (50nM, 1hr, n=6, K18 n=9) in H4i cells. All experiments in b-e were performed in biological replicates over three independent experiments and normalized to WT uptake (100%). All data expressed as mean ± s.d. One-way ANOVA with Tukey’s method, two-sided was performed to determine significance. Displayed are the comparisons against WT, ****=p-value < 0.0001. Comparison to NT sgRNA resulted in the same level of statistical significance.

Figure 2:. Tau interacts with LRP1 similar to known ligands.

a, Uptake of K18, 2N4R tau, and transferrin (Tfn) in the presence of increasing RAP concentrations in the cell media. b, Uptake of K18 and lysine capped K18 in WT H4 cells c, Schematic of LRP1 ectodomains. d, Uptake of 2N4R tau with mLRP ectodomain expression (n=9, WT vs. NT; p-value = 0.2232, WT vs. mLRP4; p-value = 0.5450). e, Uptake of K18 with mLRP ectodomain expression (n=12, WT vs. NT; p-value = 0.2648, WT vs. mLRP4; p-value = 0.0006). f, Uptake of N-terminus of tau with mLRP ectodomain expression (n=12, WT vs NT; p-value = 0.9713, WT vs. mLRP2; p-value = 0.1289, WT vs. mLRP4; p-value = >0.9999). Experiments in a-b were performed in biological duplicates over three independent experiments (n=6) and representative examples are shown. Experiments in d-f were performed in biological triplicates over at least three independent experiments and normalized to WT uptake (100%, indicated with line). All data expressed as mean ± s.d. One-way ANOVA with Tukey’s method, two-sided was performed to determine significance. Displayed is the multiple comparison against WT, ns=not-significant, ***=p-value ≤0.001, ****=p-value < 0.0001. Comparison to NT sgRNA resulted in the same level of statistical significance.

Figure 3:. LRP1 mediates tau uptake in neurons.

a, Representative images of WT or LRP1 sgRNA iPSN after tau uptake (green, Tau-488; red, neuronal marker Tuj-1; blue, DAPI; scale bar 20μm). b, Uptake of 2N4R monomers and oligomers in CRISPRi iPSN (n=10). c, Uptake of 2N4R tau (WT vs. mtRAP; p-value = >0.9999) or Tfn (ANOVA; p-value = 0.7815) in the presence of RAP or mtRAP (n=7). All experiments in a-c were performed over three independent experiments and normalized to WT uptake (100%). Data expressed as mean ± s.d. One-way ANOVA with Tukey’s method, two-sided was performed to determine significance. Displayed is the multiple comparison against WT, ns=not-significant, ****=p-value < 0.0001. Comparison to NT sgRNA resulted in the same level of statistical significance.

Figure 4:. LRP1 knockdown reduces tau spread in vivo.

a, Diagram of AAV used to study tau spread in vivo. b, Representative mosaic of a scramble shRNA injected mouse and LRP1 shRNA injected mouse (green, GFP; red, shRNA; white, hTau, scale bar 1mm) c, hTau spread in either scramble or LRP1 shRNA mice, insets represent false color densitometric maps of hTau staining d, Quantification of total number of hTau⁺/GFP^- cells/mm² of PBS, scramble shRNA or LRP1 shRNA mice (PBS vs. scramble shRNA; p-value = 0.1582). e, Quantification of total number of GFP⁺ cells/mm² (ANOVA; p-value = 0.8007) f, Quantification of ipsilateral hippocampal hTau⁺/GFP^- cells/mm² in PBS, scramble shRNA or LRP1 shRNA mice (ANOVA; p-value = 0.0692) g, Quantification of ipsilateral cortical hTau⁺/GFP^- cells/mm² in PBS, scramble shRNA or LRP1 shRNA mice (PBS vs. scramble shRNA; p-value = 0.0548). h, Image of MC-1 positive tau in PBS injected animal (green, Iba1; red, MC-1; blue, Hoechst; scale bar 20μm) i, Representative images of ipsilateral cortex from PBS, scramble shRNA or LRP1 shRNA mice (white, hTau; scale bar 20μm). j, Representative images of contralateral hippocampus from PBS, scramble shRNA or LRP1 shRNA mice (white, hTau; scale bar 20μm). Experiments in b-j n=7 animals for each group (PBS, scramble shRNA, LRP1 shRNA). Data is shown as box-plots with the median displayed as a line and the whiskers representing min/max. One-way ANOVA with Tukey’s method, two-sided was performed to determine significance, ns=not-significant, ***=p-value ≤0.001, ****=p-value < 0.0001.