Recombinant Protein Expression and Purification
Full-length human microtubule-associated protein tau was subcloned into pETM-13 (https://www.embl.de/pepcore/pepcore_services/strains_vectors/vectors/bacterial_expression_vectors/) (0N4R isoform described in Morozova et al. (2013)), and GSK-3β (a gift of the Woodgett lab, RRID:Addgene_15898) was subcloned into pBAD (EMBL). Both plasmids were generated using ligation-independent cloning (Flanagan et al. 1997). A triple tau mutant (VPR) containing P243L, V279M, and V348W in the wild-type 0N4R tau isoform was created using site-directed mutagenesis (GeneArt® Site-Directed Mutagenesis System, Thermo Fisher Scientific). P243L, V279M, and V348W in 0N4R correspond to the P301L, V337M, and R406W positions in the full-length 2N4R. The oligonucleotide sequences were as follows: P243L (CCG − > CTG): 5′-GATAATATCAAACACGTCCTGGGAGGCGGCAGTG-3′, V279M (GTG − > ATG): 5′-CAGGAGGTGGCCAGATGGAAGTAAAATCTGAG-3′, and V348W (CGG − > TGG): 5′-GACACGTCTCCATGGCATCTCAGCAATGTCTCC-3′. For expressing wild-type 0N4R and tau variant proteins, the pETM-13-0N4R tau plasmid was transformed by heat shock into chemically competent Rosetta™(DE3) cells (MilliporeSigma, 70,954) using standard techniques (Gómez-Ramos et al. 2008), and selected with 34 µg/mL chloramphenicol and 50 μg/mL kanamycin. For expressing phosphorylated tau, the pETM-13-0N4R tau plasmid was co-transformed with the pBAD-GSK3β plasmid into Rosetta-DE3 cells, and in addition to chloramphenicol and kanamycin, 100 µg/mL ampicillin was added for pBAD-GSK3β plasmid selection. Positive clones containing 0N4R tau were grown in LB media containing 34 µg/mL chloramphenicol and 50 µg/mL kanamycin, and if expressing phospho-tau with 100 µg/mL ampicillin to maintain the GSK-3β plasmid. For protein production, cells were inoculated and grown in Terrific Broth (24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol) at 37 ℃ until the A600 was between 0.5 and 0.9. To induce tau expression, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the culture was grown for 4 h at 37 °C. For phospho-tau expression, the cells co-transformed with pETM-13-0N4R and pBAD-GSK3β also received arabinose at a final concentration of 0.1%, added at the same time as the IPTG. The bacterial cells were pelleted by centrifugation at 10,000 × g and tau directly purified, or the pellet frozen at − 80 °C prior to purification.
Tau proteins were purified as described previously (Gómez-Ramos et al. 2009). Briefly, pelleted cells were resuspended in a high-salt buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M NaCl, 0.02 M NaF, 1 mM PMSF, pH 7.0), disrupted via sonication, and centrifuged at 3200 × g to remove insoluble debris. The supernatant was decanted and then boiled for 10 min, cooled on ice for 15 min, and then centrifuged at 3200 × g for 10 min to remove and discard precipitated protein. The supernatant was decanted and dialyzed overnight at 4 °C in column wash buffer (20 mM PIPES, 10 mM NaCl, 1 mM EGTA, 1 mM MgSO4, 2 mM DTT, 0.1 mM PMSF, pH 6.5). The dialyzed lysate was applied to an SP-sepharose cation-exchange column on a BioLogic DuoFlow chromatography system (Bio-Rad); following application, the column was washed for 10 column volumes and protein eluted with 0.4 M NaCl. Protein-containing fractions were combined, buffer-exchanged into PBS, and concentrated using AmiconⓇ Pro purification concentrators (10 kDa MWCO, ACS501002, MilliporeSigma). The protein concentration was determined from measuring the absorbance at 280 nm (A280) using Beer’s Law with ϵ = 7450 M−1 cm−1 and MW = 40 kDa. A typical final yield was 10–12 mg of tau per liter of culture.
SH-SY5Y neuroblastoma cells (ATCC CRL-2266™; RRID:CVCL_0019) were cultured in a 1:1 mixture of HAMS and Minimal Essential Media, containing 10% fetal bovine serum (FBS). C6 glioma cells (ATCC CCL-107™; RRID:CVCL_0194) were cultured in HAMS and 15% horse serum (ATCC) and 2.5% FBS. HEK293 cells (ATCC CRL-1573™; RRID:CVCL_0045) were cultured in DMEM and 10% FBS. CHO745 cells and CHO cells (ATCC CRL-9618™; RRID:CVCL_0214) were cultured in 10% FBS and HAMS (ATCC). Rat primary astrocytes (iXCells Biotechnologies 10RA-005) were cultured in astrocyte medium (Thermo Fisher Scientific, USA), 2% FBS and 100 U/mL Penicillin–Streptomycin. Cell cultures were maintained in a humidified atmosphere of 5% CO2 at 37 °C and cultured in CELLSTAR 25-cm2 culture flasks with filters.
CRISPR Plasmid Design and Construction
Primers of single-guide RNAs (sgRNAs) were designed with Benchling online CRISPR Guide RNA Design Tool (https://www.benchling.com/crispr/) that designs sgRNAs with input target sites and provides on- and off-target scores for optimizing higher activity and lowering off-target effects. sgRNAs were designed to target the xylosyltransferase domain; the Exon7-targeting sequence (CCTGTATGGCAACTATCCTG) on the Xylt1 gene (Acc:620,093) was introduced into the pSpCas9(BB)-2A-GFP (RRID:Addgene_48138, a gift of Feng Zhang) using established cloning protocols with BbsI and T7 ligase. The ligation reaction was transformed into Escherichia coli DH5α and the sgRNA expression plasmid obtained via selection of colonies on ampicillin-containing LB plates. Sequences were confirmed using the U6 promoter forward primer: GAGGGCCTATTTCCCATGATT (Integrated DNA Technologies, USA).
Twenty-four hours before transfection, C6 cells were seeded at 7 × 105 cells per well in a 6-well plate containing 2 mL of complete growth medium. Cells were grown overnight to approximately 80–90% confluency. On the day of transfection, cells were harvested with 0.05% Trypsin–EDTA and transfected with the sequence-confirmed sgRNA plasmid constructs. For optimizing transfection conditions, 1 × 106 C6 cells were transfected with 10 μg DNA using the SF Cell line 4D-Nucleofector™X kit (V4XC-2012, Lonza) according to the manufacturer’s specifications. Cells were incubated in a humidified 37 °C, 5% CO2 incubator for 48 h.
Fluorescence-Activated Cell Sorting (FACS) of Transfected Cells for Nuclease-Expressing Cells
Two days post-transfection, C6 cell samples were detached with 0.05% Trypsin–EDTA and the top 3% of GFP positive cells were sorted by FACS into prepared 1.5-mL Eppendorf tubes to enrich the cell population with higher levels of nuclease expression. Following a 1-week expansion, enriched cell populations were subjected to a second round of transfection with the sgRNA expression plasmid to improve the level of genome editing, by following the same procedure. Two days post-transfection, transfected cells were harvested and sorted individually into 96-well plates, followed by a 2-week expansion in a humidified incubator. The single-cell clone (CL5) used for experimentation was selected by indel detection by amplicon analysis (IDAA) DNA capillary electrophoresis (Yang et al. 2015).
Alexa Fluor Labeling
Different isoforms of tau protein were labeled with either Alexa Fluor™ 488 (AF488) NHS Ester or Alexa Fluor™ 647 (AF647) NHS Ester (Life Technologies, USA) according to the manufacturer’s instruction by incubating the label and protein (1:10 w/w) in sodium bicarbonate (pH 8.3) for 1 h at room temperature. A Sephadex G-25 column (GE Healthcare) was used to separate labeled protein from the free label and the recovered labeled protein — referred to as tau-AF488 or tau-AF647 — was concentrated using AmiconⓇ Pro purification concentrators (10 kDa MWCO) and quantified via A280 measurement prior to the use.
Live Cell Confocal Microscopy
All cells were plated at 50,000 cells/well in Lab-Tek II confocal imaging chambers (Thermo-Fisher, cat# 155,360) and cultured for 24–48 h in incubators prior to assay. On the day of assay, cell chambers were transferred to a pre-warmed live-cell imaging chamber slide incubator on a Nikon A1 confocal microscope and allowed to equilibrate for approximately 10 min at 37 °C and 5% CO2. All data acquisition was performed with a 488-nM excitation laser with a 60X oil objective attached to a heating element to prevent heat sink from the imaging chamber during acquisition. Thirty seconds after acquisition began, a pre-warmed cell culture media containing tau-AF488 was added to cells in a 2X concentration to ensure complete mixing and to minimize cell surface adherence. For analysis, individual cells were identified at each time point, and the number of tau-containing vesicles was manually counted, using the Nikon Elements software to ensure that the signal did not reach saturation. Three independent biological replicates were performed for all measurements and at least three cells per time point were analyzed. Several alternative methods to quantify tau levels (e.g., total fluorescence, first appearance of vesicles) were examined, and all gave consistent results. Due to cell adherence challenges that caused out of focus changes with time, automated measurements were not able to yield reliable values. To block non-specific endocytosis, cells were pre-incubated on loosely packed wet ice for 30 min. For experiments blocking macropinocytosis via chemical means, culture media was replaced fully with either 1.0 or 0.1 μM cytochalasin-D (MilliporeSigma C8273) for 30 min prior to tau addition to cells.
Tau Internalization Assay and Flow Cytometry
To test the tau uptake propensity by different cell types (HEK293, SH-SY5Y, C6 glioma), single-cell C6 subclone (CL5), LRP1 knockdown C6 cells, and primary rat astrocytes (RA) were seeded at 5 × 105 cells/well in a 24-well plate. After culturing overnight, cells were incubated with tau-AF488 at fixed incremental concentrations of tau proteins (0.25 μM, 0.5 μM, and 1.0 μM). After at least 30 min, the cells were washed with PBS, trypsinized with 0.05% Trypsin–EDTA (Thermo Fisher Scientific, USA), and the fluorescence analyzed with an Accuri flow cytometer (MBIC, Mellon Institute). The relative tau uptake for each condition was calculated by subtracting the non-specific binding of the fluorophore to the cell membrane, which was conducted with identical experimental procedures but at 4 ℃ to limit internalization. For some experiments, C6 glioma, CL5, or primary rat astrocytes (RA) were incubated with unlabeled tau proteins — wild-type 0N4R (tau), phospho-0N4R (ptau), and a triple 0N4R mutant (VPR) — at 1 μM for 48 h before characterization by Western blotting and quantitative real-time PCR analysis. For experiments to investigate the effects of heparin on tau uptake by cells, 0.5 mg/mL heparin (Amsbio, AMS.HEP001) along with the tau protein was added to cell culture, as indicated.
For analysis of expression or phosphorylation of different proteins present in mammalian cells, the cells were trypsinized and washed with ice-cold PBS prior to suspension in RIPA lysis buffer containing 150 mM NaCl, 0.1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mM Tri-HCL, and Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, USA) for protein extraction. The cell lysate was maintained at constant agitation for 30 min at 4 °C followed by centrifugation at 16,000 × g for 20 min at 4 °C. Protein concentration was determined by BCA assay (Thermo Fisher Scientific, USA). Prior to gel electrophoresis, cell lysate was mixed with a 4X Laemmli sample loading buffer (Bio-Rad, USA) and boiled at 95 °C for 5 min. Equal amounts of protein were loaded into the wells of a Mini-PROTEAN TGX Precast Gel (Bio-Rad, USA), along with Precision Plus Protein™ WesternC™ standards (Bio-Rad, USA) and then electrophoresed for 30 min at 225 V. Proteins were then transferred to 0.2-µm preassembled PVDF membrane (Bio-Rad, USA) using the Trans-Blot Turbo Transfer System (Bio-Rad, USA). The blots were then subjected to Western analysis as described previously (Bejoy et al. 2020). Briefly, blots were incubated with 5% nonfat dried milk in 1X Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T) buffer for 1 h at room temperature to block non-specific binding, and incubated with the primary antibodies phospho-ERK1/2 (Cell Signaling, 9101S), ERK1/2 (Cell Signaling, 9102S), Xylt1 (Life Technologies, PA5-67,627), LRP1 (Cell Signaling, 64099S), and endogenous control protein, β-actin (Cell Signaling, 3700 T), in TBS-T containing 5% (w/v) BSA overnight at 4 °C. After washing with 1X TBS-T, the blot was incubated with the fluorophore-conjugated secondary antibody (Li-Cor Biosciences, 926–32,211) for 1 h at room temperature. The blots were imaged with ChemiDoc MP Imaging System (Bio-Rad, USA) and the band intensities of target proteins were normalized to the endogenous control protein, β-actin, using ImageJ software.
Quantitative Real-Time PCR Analysis
Total RNA was isolated from cell samples using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol, followed by treatment with the RNA Clean & Concentrator Kit (Zymo). Reverse transcription of 2 μg of total RNA was carried out using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the protocol of the manufacturer. Primers used in the real-time PCR are listed in Supplementary Table 1. The gene β-actin was used as an endogenous control for the normalization of expression levels. Real-time PCR reactions were performed on a ViiA™ 7 System using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems). The amplification reactions were performed as follows: 15 min at 50 °C, 10 min at 95 °C, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Fold change in gene expression was quantified by means of the comparative Ct method, which is based on the comparison of the target gene expression (normalized to the endogenous control β-actin) between the samples. For experiments designed to inhibit MEK1/2-ERK1/2 signaling, 100 μM MEK1/2-ERK1/2 inhibitor, U0126-EtOH (Selleckchem, S1102), was added to culture media along with tau protein.
Cell staining was performed as described previously (Song et al. 2019). Briefly, 1 × 106 cells were seeded into 6 well-plate per sample and cultured overnight. The replated cells were then fixed with 4% (w/v) paraformaldehyde in PBS at room temperature for 15 min and washed twice with a staining buffer (2% (v/v) FBS in PBS). For staining intracellular markers, the samples were permeabilized with 0.2–0.5% (v/v) Triton X-100 at room temperature for 15 min. The cells were then blocked with 10% (v/v) FBS at room temperature for 30 min and then incubated with primary antibodies, mouse anti-Heparan Sulfate (HS; USBiologic, H1890), or mouse anti-Glial Fibrillary Acidic Protein (GFAP; Sigma-Aldrich, MAB360-25UL) at room temperature for 4 h. After washing, the cells were incubated with the corresponding secondary antibody: Alexa Fluor® 488 goat anti-Mouse IgM (Abcam, ab150121) and Alexa Fluor® 488 goat anti-Mouse IgG1 (Life Technologies, A21125), at room temperature for 1 h. The samples were counterstained with Hoechst 33,342 (Abcam, ab228551) and visualized using a fluorescent microscope (Keyence BZ-X810).
LRP1 Knockdown by Small Interfering RNA (siRNA)
Double-stranded, rat LRP1 specific siRNAs and non-targeting siRNA were synthesized by IDT. The sequences of LRP1 specific siRNAs were as follows:
LRP1 siRNA1: 5′-GGCGUCACUUACAUCAACAACCGTG-3′,
LRP1 siRNA2: 5′-CCUGAUGUUCUGGACCAAUUGGAAT-3′, and.
LRP1 siRNA3: 5′-GUAAAAAUGAAGGAAUUACUUUUTA-3′.
The 1 × 106 C6 glioma cells were transfected with LRP1-specific siRNAs (300 nM) and non-targeting siRNA using Lonza’s SF Cell line 4D-Nucleofector™X kit (V4XC-2012, Lonza) according to the manufacturer’s specifications. Cells were incubated in a humidified 37 °C, 5% CO2 incubator for 48 h. Gene expression of LRP1 was analyzed by real-time PCR analysis 48 h after transfection.
Statistical significance was determined by unpaired t-tests, one-way analysis of variance (ANOVA), or two-way ANOVA when appropriate using GraphPad Prism 8 software. A 5% cutoff was applied to determine statistical significance, and a p-value of < 0.05 was denoted with one asterisk (*). In all flow cytometry and imaging experiments, at least three independent biological replicates from different days were performed and the standard error of the mean is shown. In all cases, the number of replicates is included in the figure caption.