LRP1 is a master regulator of tau uptake and spread

Summary The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE)1. 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 studies3,4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation.

Recent work has highlighted the importance of heparan sulfate proteoglycans (HSPGs) in tau uptake 5,6 , and low-density lipoprotein receptors (LDLRs) are known to work in conjunction with HSPGs 7 . On the basis of this knowledge, we set out to test whether any members of the LDLR family could influence tau internalization. Using CRISPR interference (CRISPRi) technology, the expression of genes encoding various members of the LDLR family (LRP1, LRP1B, LRP2, LRP5, LRP8, LDLR and VLDLR; Extended Data Fig. 1c) was efficiently repressed in H4 neuroglioma cells, and the ability of these cells to endocytose monomeric tau was assessed 5 (Fig. 1a, b). Genetic silencing of LRP1 almost completely blocked the uptake of full-length soluble monomeric tau (2N4R isoform), whereas no significant effect was found upon silencing any other LDLR family member (Fig. 1b). Different mechanisms have been proposed for the uptake of soluble and of aggregated tau 8 . However, LRP1 knockdown was also sufficient to inhibit the uptake of tau oligomers, and reduced but did not completely inhibit the uptake of sonicated tau fibrils (Fig. 1c). The uptake of a disease-relevant mutant of tau and of phosphorylated tau was also affected by LRP1 knockdown (Extended Data Fig. 1d). To show that LRP1 knockdown was specific for tau endocytosis, we analysed the uptake of transferrin in H4 cells; LRP1 knockdown was found to have no effect on transferrin endocytosis (Fig. 1d). Knockdown of LRP1 also prevented uptake of the smaller isoforms of tau (0N3R, 0N4R, 1N3R, 1N4R and 2N3R) as well as fragments of tau that contain only the microtubule-binding repeat region (K18, 4 repeats; K19, 3 repeats; Fig. 1e). These findings highlight the microtubule-binding region as the potential interaction site.
To further support the results from the LRP1-knockdown cell lines, a well-known LRP1-binding protein-receptor-associated protein (RAP)-was used as a competitor for tau uptake in wild-type H4 cells. RAP is a small, 39 kDa chaperone for LRP1 that is known to bind tightly to LRP1 (dissociation constant, K D = 9 nM) 9 . Increasing concentrations of RAP in the culture medium, added concurrently with tau, were highly effective at inhibiting the uptake of both full-length tau and K18 tau (half-maximum inhibitory concentration, IC 50 = 4.9 nM and 9.6 nM, respectively); however, they did not influence the internalization of transferrin (Fig. 2a). We next introduced point mutations at residues of RAP that are required for its interaction with LRP1 10 ; the mutation of lysines 256 and 270 to alanine (to form the mutant RAP(K256A/K270A), hereafter denoted mtRAP) was sufficient to reduce this competitive effect (Extended Data Fig. 2a, b).
In an attempt to understand how tau may interact with LRP1, we examined known crystal structures of members of the LDLR family bound to their ligands 11,12 . LRP1 (and other members of the LDLR family) contain cysteine-rich complement-type repeats that bind and internalize their Article ligands. Each complement-type repeat is composed of approximately 40 amino acids: six cysteines and a cluster of acidic residues (normally aspartic acid) that coordinates Ca 2+ and interacts with lysine residues on ligands through salt bridges 13 . Tau has a high lysine content-out of 441 amino acids, 44 (about 10%) are lysines, 20 of which are located within the microtubule-binding region (the lysine content of the K18 fragment is around 15%). Furthermore, cryo-electron microscopy structures of tau fibrillar aggregates from the brains of patients with Alzheimer's disease or chronic traumatic encephalopathy 14,15 show 10 or 11 resolved lysines, with all but one exposed to the exterior and therefore available for interactions with other proteins. To assess whether salt bridges between tau lysines and LRP1 were necessary for tau uptake, we capped all lysine residues on the K18 fragment using a sulfo-NHS acetate (NHS, N-hydroxysuccinimide) and measured endocytosis. The capping of lysine residues on K18 prevented the uptake of tau in wild-type H4 cells (Fig. 2b), which indicates that-similar to other LRP1 ligands-lysine residues are critical for this interaction.
LRP1 is a large (600 kDa) member of the LDLR family, and it contains 31 complement-type repeats divided into four different ligand-binding domains 16 . To elucidate which of these ligand-binding domains influences tau uptake, we designed ectodomain constructs-known as mini-LRPs (mLRPs)-to assess the ability of individual subdomains (Fig. 2c) to rescue tau internalization in our LRP1-knockdown cells. By expressing these mLRPs in the absence of full-length LRP1 (Extended Data Fig. 2c), we found that full-length tau uptake was completely rescued with subdomain 4 (mLRP4) and partially rescued with mLRP2 ( Fig. 2d). Furthermore, mLRP4 was able to co-immunoprecipitate tau, which confirms an interaction between the two (Extended Data Fig. 2d). Only mLRP4 improved K18 uptake (Fig. 2e), indicating that two motifs in tau could possess the ability to bind LRP1: one within the microtubule-binding region (amino acids 244-372) that interacts with mLRP4, and one in the N-terminal half (amino acids 1-243) or the C-terminal end (amino acids 373-441) that interacts with mLRP2. To test this further we examined the uptake of the N terminus of tau (amino acids 1-243), and found that both mLRP4 and (to a lesser extent) mLRP2 could rescue N-terminal uptake (Fig. 2f). This suggests a model in which a primary interaction site on mLRP4 mediates tau uptake, but that the N terminus of tau can also mediate interactions with a secondary site on mLRP2. We also noted that, although LRP1 knockdown was sufficient to reduce uptake of the N terminus of tau, we did not observe the marked inhibition that was seen with constructs containing the microtubule-binding repeat region. This might be attributed to the higher concentrations of the N terminus that are required under our experimental conditions (Extended Data Fig. 2e), and could suggest that there is a secondary route of tau endocytosis, mediated by a slower, LRP1-independent pathway. To determine whether other known ligands of mLRP2 and mLRP4 are able to compete for uptake, we overexpressed various ApoE isoforms in HEK293T cells and collected the conditioned medium containing ApoE (Extended Data Fig. 2f). The results showed that incubation of H4 cells in ApoE conditioned medium significantly reduced the uptake of full-length tau, yet failed to reduce transferrin uptake (Extended Data Fig. 2g).
Native LRP1 is highly expressed in neurons at the post-synaptic density 17,18 , and previous work has highlighted that the spread of tau in vivo is probably mediated trans-synaptically. Therefore, we asked whether the uptake of tau in neurons was also regulated by LRP1. We reduced the expression of LRP1 in human induced pluripotent stem cell-derived neurons (iPSNs) using CRISPRi (Extended Data Fig. 3a). Micrographs of adherent iPSNs showed that LRP1 knockdown efficiently reduced the amount of internalized tau (Tau-488, green; Fig. 3a). Quantification of tau endocytosis by flow cytometry confirmed the reduction of tau uptake upon LRP1 knockdown, or upon the addition of RAP-but not mtRAP-into the culture medium (Fig. 3b, c). Similar to the results in H4 cells, LRP1 knockdown or the addition of RAP had no effect on transferrin uptake in iPSNs (Fig. 3c).
On the basis of these in vitro results, we sought to determine whether LRP1 was also critical for the spread of tau in the brain. A recently described model of tau spread uses an adeno-associated virus (AAV) and enables reliable discrimination between neurons that have been transduced to express human tau (hTau) and neurons that receive hTau through spread 19 . The AAV encodes one mRNA-GFP-P2A-hTau under the control of a cytomegalovirus promoter-but produces two proteins, GFP and hTau (Fig. 4a). The P2A peptide selfcleaves during translation 20 -as such, transduced neurons can be identified by the presence of GFP and hTau, whereas cells that have taken up tau via spread mechanisms can be identified by the absence of GFP and the presence of hTau (Fig. 4a). To regulate the expression of LRP1 in parallel, an AAV (PHP.eB serotype) carrying a short hairpin RNA (shRNA) targeting LRP1 under the human synapsin promoter was used to knock down LRP1 in neurons (Extended Data Fig. 3b, d). We administered AAVs encoding either the shRNA targeting LRP1 or a control 'scramble' shRNA through retro-orbital injection into six-week-old wild-type mice, and two weeks later we administered stereotactic injections of the AAV GFP-P2A-hTau virus into the hippocampus. After three weeks of incubation, the mice were euthanized One-way ANOVA with Tukey's method, two-sided was performed to determine significance. Displayed are the comparisons against the wild type; ****P < 0.0001. Comparison with NT sgRNA resulted in the same level of statistical significance. and spread was determined by immunofluorescence experiments (Fig. 4b, c). AAVs encoding scramble shRNA and shRNA against LRP1 contained the fluorescent protein reporter mRuby, which enabled us to visualize their expression throughout the brain (Fig. 4b). Mice expressing shRNA against LRP1 showed reduced expression for LRP1, as determined by quantitative PCR and immunohistochemistry analysis (Extended Data Fig. 3b, f). To quantify tau spread, the number of hTau + /GFP − cells was counted after immunostaining for human tau (Fig. 4c, d). We observed a substantial amount of tau spread in mice injected with PBS and scramble shRNA (mean ± s.d. 199 ± 73 and 147 ± 43 hTau + /GFP − cells per mm 2 , respectively), whereas spread was greatly diminished in LRP1-knockdown mice (19 ± 19 hTau + /GFP − cells per mm 2 ) (Fig. 4d). This was not due to differences in the number of transduced cells, as we observed equivalent amounts of GFP + cells across all mice (Fig. 4e). When analysing tau spread by brain region, we found that whereas mice injected with shRNA against LRP1 had similar numbers of spread cells in the ipsilateral hippocampus ( Fig. 4f), spread was significantly diminished in the cortex (Fig. 4g, i).
We observed no effect of sex on the spread phenotype (Extended Data Fig. 3c). LRP1 is highly expressed in neurons, but it has also been shown to be present in other cell types including astrocytes and microglia 21 . Although not a focus of this work, we did observe instances of hTau + astrocytes (Extended Data Fig. 3e); this will be a topic of future studies. Consistent with previous reports 19 , we found examples of MC-1 (an antibody recognizing an altered conformation of tau) reactivity in mice injected with PBS and scramble shRNA (Fig. 4h). Tau spread was also observed in the contralateral hippocampus of such mice, but was absent in mice injected with shRNA against LRP1 (Fig. 4j). Our study identifies LRP1 as a master regulator of tau protein endocytosis in neurons, with an important role in the spread of tau in the brain. Targeting neuronal LRP1 led to a significant reduction of tau spread in vivo, and could point to a new therapeutic approach for tau-related neurodegenerative diseases. The importance of LRP1 in Alzheimer's disease has previously been attributed to its regulatory role in both the degradation and production of amyloid-β, and the internalization of ApoE 22 . ApoE4 has been shown to exacerbate amyloid-β pathology in a manner that depends on LRP1 expression 23 , and has also been found to worsen tau pathology 24 . ApoE4 is known to have the lowest levels of circulating protein in the brain relative to the other ApoE isoforms 25 , which suggests that it would be the least effective at inhibiting tau-LRP1 interactions and, therefore, at suppressing tau spread. Together, our results point to LRP1 as a common factor that can help to explain the co-occurrence of amyloid and tau lesions. Recently, tau uptake studies have focused on mechanisms dependent on HSPGs 5,6 . However, HSPGs are known to act both independently and together with coreceptors to mediate endocytosis 26 . This work therefore improves our understanding of tau spread, highlights LRP1 as a critical determinant were performed as biological triplicates over at least three independent experiments and normalized to wild-type uptake (100%, indicated by lines). All data are expressed as mean ± s.d. with individual data points shown. One-way ANOVA with Tukey's method, two-sided was performed to determine significance. Displayed is the multiple comparison against the wild type; NS, not significant, ***P ≤ 0.001, ****P < 0.0001. Comparison with NT sgRNA resulted in the same level of statistical significance. . All experiments in a-c were performed over three independent experiments and normalized to wild-type uptake (100%). Data are expressed as mean ± s.d. with individual data points shown.
One-way ANOVA with Tukey's method, two-sided was performed to determine significance. Displayed is the multiple comparison against the wild type; NS, not significant; ****P < 0.0001. Comparison with NT sgRNA resulted in the same level of statistical significance.

Article
for tau propagation and suggests that interventions along a single pathway or at a single nidus-in which tau, ApoE and HSPGs operate dynamically in conjunction with LRP1-may remediate both plaque and tangle pathologies.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-020-2156-5.  One-way ANOVA with Tukey's method, two-sided was performed to determine significance; NS, not significant; ***P ≤ 0.001, ****P < 0.0001.

Protein purification and labelling
Tau proteins were purified as described previously 5 . RAP, mtRAP, and the N terminus of tau were cloned into the pMCSG7 and purified on nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen). The His-tag was removed by tobacco etch virus (TEV) protease cleavage and subsequent clean-up on Ni-NTA agarose. Transferrin was purchased from Millipore and labelled in-house. Protein was labelled with Alexa Fluor 488 or 647 5-SDP ester (Life Technologies) according to the supplier's instructions. After labelling, 100 mM glycine was added to quench the reaction and the proteins were subjected to Zeba desalting columns (Thermo Scientific) to remove any unreacted label. Average label incorporation was between 1 and 1.5 moles per mole of protein, as determined by measuring fluorescence and protein concentration (A max × molecular mass of protein / [protein] × ε dye , where A max is the absorbance measured at the maximum absorption wavelength and ε dye is the molar extinction coefficient of the dye). Lysine capped proteins were prepared with Sulfo-NHS-Acetate (Thermo) according to the manufacturer's instructions. Phosphorylation of tau was achieved by incubation with mouse brain extract in accordance with published work 29 and confirmed via western blotting. To prepare tau fibrils and oligomers, 10 uM protein, in PBS 1 mM DTT pH 7.4, was mixed with heparin (0.05 mg ml −1 ) and incubated with shaking at 37 °C. The formation of oligomers was observed after 4 h of shaking, whereas fibril formation occurred after 5 days as previously described 30 . To make sonicated fibril samples, fibrillized protein was sonicated using a Model 120 Sonicator (Fisher) at 30% amplitude for 30 × 1 s pulses.
Tau uptake assay H4 cells were plated at 50,000 cells per well and iPSN were plated at 100,000 cells per well in a 24-well plate. The next day the medium was replaced, and cells were treated with Alexa Fluor 488-or Alexa Fluor 647-labelled tau protein (50 nM) or Alexa Fluor 488-labelled transferrin (500 nM) for 1 h at 37 °C (unless otherwise indicated in the figure legends). Cells were then washed twice with PBS and trypsinized to lift cells from the plate. Lifted cells were analysed using an Accuri-C6 Flow Cytometer (BD Biosciences) and propidium iodide was used to exclude dead cells from the analysis (Extended Data Fig. 1a). Experiments were run in biological duplicates or triplicates and data was normalized to wild-type uptake to combine datasets from multiple experiments. Internalization controls are provided in Extended Data Fig. 1b. For RAP competition, recombinant RAP protein was added into the medium at indicated concentrations at the same time as labelled tau. For ApoE competition, ApoE-conditioned medium was used as the replacement medium before tau addition.

AAV production and purification
For shRNA, PHP.eB AAVs were generated and collected as previously described 32 . This engineered capsid enables efficient transduction of the central nervous system via the vasculature 33 . Sequences for LRP1 shRNA (CCGGGCTGAACACATTCTTTGGTAACTCGAGTTACCAAAGAA TGTGTTCAGCTTTTTG), and scramble shRNA (CCGGCAACAAGATGA AGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG) were obtained from Sigma and were tested before AAV cloning in mouse primary culture. Purified viruses were concentrated, washed in PBS, sterile-filtered and titred using quantitative PCR. EGFP-2A-hTauP301L constructs were packaged into AAV2/8 (Massachusetts Eye and Ear Institute vector core).

Mice
All procedures were performed with approval from the Institutional Animal Care and Use Committee and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and guidelines from the University of California, Santa Barbara. Mice were maintained in ventilated cages, on a standard rodent diet of chow and water ad libitum and housed under a 12-hour light/dark cycle. Experiments were performed starting at 6 weeks of age on a sexmixed (4 males and 3 females per condition) cohort of in-house bred mice (FVB/B6). Mice were injected retro-orbitally with either PBS or an AAV PHP.eB of mRuby-shRNA with either a scramble or LRP1 sequence. After two weeks, mice were intracranially injected with AAVs encoding EGFP-2A-hTauP301L into the hippocampus (1.5 μl unilaterally). Injections were performed under standard aseptic surgery conditions. Mice were anaesthetized with isoflurane (2%) and injections were made at the coordinates (from bregma A/P −2 mm, M/L −1.5 mm, D/V −1.5mm). A Hamilton needle was used to inject the AAV solution at 0.5 μl min −1 . Afterwards, the skin over the injection site was sutured and mice were allowed to recover on a warming pad. Mice received meloxicam for two days after surgery. No calculations were performed to predetermine sample size, sample sizes were based on previously published work and were chosen to support meaningful conclusions, and no specific method of randomization was used.

Immunohistochemistry
Mice were perfused using 4% paraformaldehyde in 0.1 M sodium cacodylate pH 7.4 for 15 min at room temperature. Brains were then immersion-fixed for 48 h at 4 °C. Immunocytochemistry was performed as described elsewhere 34 ; in brief, samples were rinsed 3 × 5 min, 1 × 1 h in cold phosphate buffered saline (PBS; pH: 7.4) and then sectioned at 100 μm using a vibratome (Leica). Sections were immersed in normal donkey serum ( Jackson ImmunoResearch Laboratories) 1:20 in PBS containing 0.5% BSA, 0.1% Triton-X 100, and 0.1% sodium azide (PBTA) at 4 °C on a rotator with overnight agitation. Next, sections were immersed in primary antibodies, anti- Tau13

Large-scale mosaic acquisition and image registration
Samples were imaged using an Olympus FV1000 confocal microscope equipped with a precision motorized stage (Applied Scientific Instrumentation). Mosaics were captured using an UPlanSApo 20× oil immersion lens with numerical aperture 0.85 at 1-μm intervals in the z-dimension and a pixel display of 800 × 800 along the x-y axes. Image stacks were collected sequentially using the Olympus Fluoview software v.4.2 with 5% overlap between individual tiles. Image registration of individual z-stacks was performed in a semi-automated fashion using the bio-image software Imago 1.5 (Mayachitra). False-colour densitometric maps were generated using x, y coordinates from annotated images using the visualization open source package ggplot2 version 3.2.1 as part of RStudio desktop v.1.2.5001 and subsequently rendered using R v.3.6.1. Animal mosaics were quantified under blinding conditions, file names were withheld until after image analysis was completed.

Statistical analysis
All statistical analysis was performed using Prism 8 software. To determine statistical significance, Shapiro-Wilk tests were first used to evaluate the assumption of normality of the data. Given P > 0.05, then normality assumptions were not rejected and ANOVA tests were used. In certain cases, outliers were removed to meet the normality assumption and then an ANOVA test was performed; however, all data are displayed in full. Post-hoc analysis of ANOVA was performed using Tukey's method in order to compare experimental samples to both wild-type and nontargeting sgRNA (or scramble shRNA) controls. For quantitative PCR data, unpaired t-tests were used to determine significance.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability
Source data are available for graphs plotted in Figs. 1-4 and Extended Data Figs. 1-3. Scans of the full western blot gels can be found in Supplementary Fig. 1. All other data are available from the corresponding author upon reasonable request.

Software and code
Policy information about availability of computer code Data collection BD Accuri C6 software was used to acquire flow cytometry data. Quantstudio Design and Analysis Software v1.4 was used to acquire qPCR data. Odyssey software was used to capture western blot images. Image stacks were collected sequentially using the Olympus Flouview software version 4.2 with 5% overlap between individual tiles.

Data analysis
Prism 8 was used to perform statistical analysis on all data presented. Image registration of individual z-stacks was performed in a semiautomated fashion using the bio-image software Imago 1.5 (Mayachitra Inc.).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability An excel file containing source data for the following figures has been included with submission ( Fig. 1B, 1C, 1D Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

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Life sciences study design
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Sample size
No calculations were performed to predetermine sample size, sample sizes were based on previously published work and chosen to support meaningful conclusions. The sample size (n) of each experiment is provided in the figure captions.
Data exclusions No animals or samples were excluded from the displayed datasets. To determine statistical significance, Shapiro-Wilk tests were first used to evaluate the assumption of normality of the data. Given a p>0.05 then normality assumptions were not rejected and ANOVA tests were used. In certain cases, outliers were removed to meet the normality assumption and then an ANOVA test was performed, however data is displayed in full.

nature research | reporting summary
October 2018

Mycoplasma contamination
All cell lines used tested negative for mycoplasma contamination using a PCR Mycoplasma test kit (VWR 10181-030).
Commonly misidentified lines (See ICLAC register) None used.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Experiments were performed on six-week old mice, (FVB/B6, four males/three females were used for each experimental cohort).

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve samples collected from the field.

Ethics oversight
All procedures were done with approval from the Institutional Animal Care and Use Committee and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and guidelines from the University of California, Santa Barbara.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Cells were obtained from culture conditions and were lifted and analyzed without fixation.

Instrument
BD Accuri C6 for routine analysis and Sony SH800 for sorting Software BD Accuri C6 software for collection and Prism 8 software for data analysis.
Cell population abundance Cells that were infected with mLRP constructs were sorted using a Sony SH800 Cell Sorter on EGFP expression. Cells were sorted using a standard two-way sort. Cells were determined to be >99% positive after sorting.
Gating strategy Cells were gated on FSC/SSC (Mean FSC-A: ~ 8,000,000/Mean SSC-A: ~800,000). Cells were 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. See Extended Data Fig. 1a.
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