3D human neuronal cell culture model + microglia
GFP-expressing ReNcell VM neuronal cell line G10, previously described76, was seeded as a 3D culture in a 96-well plate (Greiner Bio-One, 655866) in ReN Differentiation Media (ReN Diff). ReN Diff changes were completed twice a week until the plate was 3.5−4 weeks old, at which the neuronal culture is considered mature, fully differentiated and ready for experimentation. The 3D human triculture system including neurons, astrocytes and microglia was previously described77. In brief, induced pluripotent stem cells are differentiated into mature microglia over 38 days. Once mature, the microglia were resuspended and added at a 6:1 ratio to the 3D ReNcell VM culture for 24 hours followed by a 24-hour HSV-1 infection. After infection, the wells were washed once with PBS and then fixed with 4% paraformaldehyde (PFA) for 2 hours at 4 °C. Then, the plate was washed three times with PBS and stored in PBS at 4 °C until ready for immunostaining. Plate was first blocked with Doo Block shaking at 4 °C overnight. The next day, primary antibodies anti-IBA1 (Abcam, ab283346) at 1:1,000 and anti-p-tau (PHF1, Albert Einstein College of Medicine) at 1:500 were added and incubated at 4 °C overnight shaking. The next day, the plate was washed five times at 5-minute intervals with TBS-Tween. Fluorescent secondaries anti-rat 405 (Invitrogen, A48268) and anti-mouse 647 (Invitrogen, A32787) were added at 1:500 for a 3-hour incubation at 4 °C shaking. Afterwards, the wells were washed five times for 5 minutes each with TBS-Tween and then stored in PBS. Plates were imaged by fluorescence confocal microscopy (Nikon, A1R-HD25) for microglia (405), neurons (GFP), HSV-1 (RFP) and p-tau (647).
Tau HSV1 plaque assay
Two-dimensional (2D) ReNcell VM cultures were seeded in a 24-well plate (Corning, 3526) precoated with 1:100 Matrigel (Corning, 354234) diluted in cold DMEM F-12 at 300,000 cells per well in ReN Expansion Media (ReN Exp) overnight. The next day, the media were replaced by ReN Diff. At 1 week old, the media were removed and replaced with ReN Diff containing 2N4R GSK-3β p-tau (Abcam, ab269024), 2N3R tau (Abcam, ab72462) or 2N4R tau (Abcam, ab84700) overnight at 37 °C at the following concentrations: 2.5 μg ml−1, 1.25 μg ml−1 and 0.625 μg ml−1. ReN Diff alone was used for the control wells. The next day, the media were removed, and ReN Diff containing HSV-1 (diluted to 50−200 plaque-forming units (PFU)) were added to each of the wells and centrifuged (Thermo Fisher Scientific, ST8R centrifuge) at 500g for 1 hour at room temperature. The HSV-1 media were removed, and ReN Diff containing 0.36% agarose were added to the wells for an overnight incubation at 37 °C. The next day, the plate was imaged by confocal microscopy (Nikon, A1R-HD25) for viral RFP fluorescent signal. Images were analyzed using Nikon Elements General Analysis 3 (GA3) for viral plaque counts, HSV-1-positive single cells and viral plaque size.
GSK-3β inhibitor and anti-INFγ antibody HSV-1 plaque assays
‘Tau HSV-1 plaque assay’ methodology was adapted to be performed with combinations of a GSK-3β inhibitor (Tocris Bioscience, 4423) and an anti-INFγ antibody (Invivogen, HIFNG-MAB7-02) in place of the synthetic taus from start to finish including image analysis. GSK-3β inhibitor was used at the following concentrations: 50 μg ml−1, 25 μg ml−1 and 12.25 μg ml−1; and anti-INFγ antibody was used at 10 μg ml−1 for the overnight incubation. In addition, GSK-3β inhibitor was also mixed in with the agarose in its respective concentrations.
Viral capsid isolation
One-half percent Triton X-100 was added to 900 μl of TNE buffer and 100 μl of HSV-1 to create a virus mix stored at 4 °C for 5 minutes. TNE buffer with 35% sucrose was added to ultracentrifuge tubes (Beckman Coulter, 344059) before adding virus mix and then ultracentrifuged (Beckman Coulter, Optima XPN-100 Ultracentrifuge) at 37,000g for 45 minutes at 4 °C. After ultracentrifugation, supernatant was aspirated, and the capsid pellet was resuspended in 200 μl of TNE buffer. Capsid mix was added to TNE buffer with 35% sucrose and ultracentrifuged again at 37,000g for 45 minutes at 4 °C. The supernatant was aspirated, and capsid-isolated HSV-1 was resuspended in 200 μl of TNE buffer followed by storage at 4 °C for experimentation.
Virus binding assay
Capsid-isolated HSV-1 or whole virion HSV-1 (5 × 106 PFU per well) in distilled water (50 μl per well) was added to a black polypropylene plate (Greiner Bio-One, 655209) and heat-fixed at 95 °C for 2 hours. After heat-fixing, the plate underwent three TBS washes and then was blocked at room temperature for 1-hour shaking using 4% non-fat dry milk. After blocking, the plate was washed three times with TBS-Tween. Synthetic taus 2N4R GSK-3β p-tau (Abcam, ab269024), 2N3R tau (Abcam, ab72462), 2N4R tau (Abcam, ab84700) or 50/50 mix of 2N3R/2N4R taus were added to appropriate wells and incubated at 37 °C for 1 hour at the following concentrations: 5.0 μg ml−1, 2.5 μg ml−1, 1.25 μg ml−1, 0.625 μg ml−1 and 0.3125 μg ml−1. After tau incubation, the plate was washed three times with TBS-Tween and then blocked at room temperature for 1-hour shaking using 10% BSA. After blocking, the plate was washed three times with TBS-Tween followed by the addition of an anti-tau antibody (Agilent Technologies, A0024) at 1:10,000 in 10% BSA (100 μl per well) and incubated at 4 °C overnight shaking. The next day, the plate was washed three times with TBS-Tween followed by addition of an anti-rabbit HRP antibody (Cytiva, NA934-100UL) at 1:1,000 in 10% BSA (100 μl per well) and incubated at room temperature for 1-hour shaking. Afterwards, the plate was washed three times with TBS-Tween before adding SuperSignal ELISA Femto Substrate (Thermo Fisher Scientific, 37075). The plate was immediately read on a BioTek Synergy Neo2 Multi-Mode Reader for chemiluminescent signal.
Competitive inhibitor virus binding assay
‘Virus binding assay’ methodology was adapted to be performed with anticapsid interference antibodies prior to synthetic tau addition. The initial steps up to 4% non-fat milk block and subsequent TBS-Tween washes remain the same. After the washes, anticapsid interference antibodies anti-VP21/VP22a (Thermo Fisher Scientific, MA5-16798), anti-ICP5 (Virusys Corporation, HA018-100), anti-pUL25 (University of Pittsburgh), anti-pUL48-VP16 (Novus Biologicals, NB206593) or anti-RS1-ICP4 (Abcam, ab6514) were added at stepwise dilutions (0.04, 0.2, 1.0 and 5.0 μg ml−1) to appropriate wells and incubated at 37 °C for 1 hour. The plate was then washed three times with TBS-Tween. 2N4R GSK-3β p-tau (Abcam, ab269024), control scrambled LL-37 or 2N4R GSK-3β p-tau incubated with mannose for 1 hour at room temperature was added to wells and incubated at 37 °C for 15 minutes. Then, the plate was washed three times with TBS-Tween followed by a 10% BSA block at room temperature for 1 hour. A subsequent three-times TBS-Tween wash was performed, and a 10% BSA of anti-tau antibody (Agilent Technologies, A0024) at 1:10,000 was added to each well (100 μl per well) for an overnight incubation at 4 °C shaking. The next day, the plate was washed three times with TBS-Tween, and an anti-rabbit HRP antibody (Cytiva, NA934-100UL) at 1:1,000 in 10% BSA (100 μl per well) was added to each well for a 1-hour room temperature incubation shaking. The plate was washed three times with TBS-Tween before adding SuperSignal ELISA Femto Substrate (Thermo Fisher Scientific, 37075). The plate was immediately read on a BioTek Synergy Neo2 Multi-Mode Reader for chemiluminescent signal.
Viral agglutination and TEM analysis
Sample containing capsid-isolated HSV-1 (10 μl) and 2N4R GSK-3β p-tau (10 μl) was incubated for 2 hours at room temperature before being added (10 μl) onto Formvar/Carbon Support Square Grids (Electron Microscopy Sciences, FCF100-Cu) for 2 minutes. Sample was removed by gently touching the perimeter of the grid with filter paper. The grid was transferred to water droplets for 30 seconds three times. After washes, the grid was dyed by adding 10 μl of 1% uranyl acetate for 1 minute before removing the dye with filter paper and letting the grid sit at room temperature for 10 minutes to dry. Grids were imaged with a JEOL 1011 electron microscope (JEOL Institute).
Slice TEM analysis
3D ReNcell VM cultures were seeded in a six-well plate at 8 million cells per well in ReN Diff and were aged for 3.5 weeks. The cells were infected with HSV-1 (4.06 × 107 PFU per well) and centrifuged with minimal acceleration and deacceleration (Thermo Fisher Scientific, ST8R centrifuge) at 500g for 1 hour at room temperature and then incubated at 37 °C for 24 hours. The next day, the media were removed, and the cells were washed once with PBS followed by initial fixation in 4% PFA on a rotator for 30 minutes. Then, fresh 4% PFA was added, and the cells were incubated at 4 °C overnight. The next day, the cells were washed three times using PBS, scraped and spun at 2,200g for 15 minutes. The supernatant was removed, and the pellets were resuspended in PBS to be stored at 4 °C. Pellets were loaded onto grids and stained with anti-p-tau (PHF1) antibody incubated with immunogold particles in accordance with ‘Viral agglutination and TEM analysis’ methodology.
Electron microscopy was performed in the Microscopy Core of the Center for Systems Biology/Program in Membrane Biology, which is partially supported by an Inflammatory Bowel Disease grant (DK043351) and a Boston Area Diabetes and Endocrinology Research Center (BADERC) award (DK057521).
Total tau and p-tau level MSD
3D ReNcell VM cultures were seeded in a 96-well plate (Greiner Bio-One, 655866) and aged for 3.5−4 weeks. At maturation, half the media was removed from the wells and replaced with ReN Diff containing HSV-1 (6.67 × 105 PFU per μl) at 0.5 µl, 0.25 µl or 0.1 µl per well for 24 hours at 37 °C. The next day, the plate was washed once with cold PBS, and cold Sarkosyl lysis buffer was then added to the wells for a 1-hour incubation at 4 °C rocking. The cells were then scraped and collected in a 96-well polymerase chain reaction (PCR) plate to be stored at −20 °C. The cell lysate was thawed and spun (Thermo Fisher Scientific, ST8R centrifuge) at 2,129g for 30 minutes to pellet cellular debris. The supernatant was collected and added to an ultracentrifuge tube (Beckman Coulter, 343778) and spun at 279,000g (Beckman Coulter, Optima MAX-XP Ultracentrifuge) for 1 hour to separate insoluble and soluble tau. After ultracentrifugation, the supernatant was collected as soluble tau. The remaining pellet was dissolved using guanine hydrochloride and collected as insoluble tau. Both soluble and insoluble tau were added to a 96-well MSD Multi-Spot Phospho (Thr 231)/Total Tau Assay plate (MSD, K15121D-2) and run according to the manufacturer’s instructions. Plates were read on an MSD MESO QuickPlex SQ 120 mm, and the resulting raw data were analyzed using MSD Discovery Workbench.
Proinflammatory cytokine MSD
Media for proinflammatory cytokine concentration determination were collected from 24-well plates preincubated with 1.25 µg ml−1 2N4R GSK-3β p-tau (Abcam, ab269024) for 24 hours with or without 1-hour HSV-1 infection the next day. Conditioned media were added to the 96-well V-PLEX Proinflammatory Panel 1 Human Kit (MSD, K15049D) and run according to the manufacturer’s instructions. Plates were read on an MSD MESO QuickPlex SQ 120 mm, and the resulting raw data were analyzed using MSD Discovery Workbench.
P-tau and total tau immunofluorescence labeling
3D ReNcell VM cultures were seeded in a 96-well plate (Greiner Bio-One, 655866) and aged for 3.5−4 weeks. At maturation, the plate was infected with 30 μl of replication-deficient HSV-1 virus (3.3 × 106 PFU per μl; Heming et al. 2013) mixed with 6 ml of ReN Diff or HSV-1−RFP fusion virus and incubated at 37 °C for 24 hours. The wells were then washed once with PBS and then fixed with 4% PFA at 4 °C for 2 hours. Afterwards, the plate was PBS washed three times followed by blocking with Doo Block at 4 °C shaking overnight. The next day, the following primary antibodies were added for an overnight 4 °C incubation shaking: anti-total tau (Agilent Technologies, A0024) at 1:1,000, anti-p-taus PHF1 (Albert Einstein College of Medicine, gift) or AT180 (Invitrogen, MN1040) at 1:500 and anti-HSV-1 (Invitrogen, PA5-115473) at 1:1,000 only if the replication-deficient HSV-1 was used. The next day, the plate was washed five times at 5-minute intervals using TBS-Tween, and the following secondary antibodies (1:500) were added for a 3-hour 4 °C incubation shaking: anti-rabbit 405 (Jackson ImmunoResearch, 711-475-152), anti-mouse 647 (Invitrogen, A32787) and anti-rabbit 568 (Invitrogen, A10042) if necessary. Afterwards, another five 5-minute TBS-Tween washes were repeated, and the plate was stored in PBS. Plates were imaged by fluorescence confocal microscopy (Nikon, A1R-HD25) and analyzed using Nikon Elements GA378.
Microfluidic immunofluorescence
Polydimethylsiloxane microfluidic devices were designed and produced at the Center for Engineering in Medicine at Massachusetts General Hospital (MGH), as described previously77. The devices were precoated for 3 hours with 1:100 Matrigel (Corning, 354234) diluted in cold DMEM F-12. 2D ReNcell VM cultures were seeded at 300,000 cells for the outside chambers and 150,000 cells for the middle chamber. The devices were aged for 3.5 weeks followed by a 48-hour 0.5 μl per well HSV-1 (6.67 × 105 PFU per μl) infection of the left chamber. After a PBS wash, the devices were fixed with 4% PFA for 2 hours at 4 °C. The devices followed the immunostaining ‘p-tau and total tau immunofluorescence labeling’ methodology with a distinction in antibody use. Instead, the antibodies anti-p-tau at 1:500 (PHF1, Albert Einstein College of Medicine) and anti-total tau at 1:750 (GeneTex, GTX49353) fluorescently labeled with secondaries anti-chicken 405 at 1:2,000 (Jackson ImmunoResearch, 703-475-155) and anti-mouse 647 at 1:1,000 (Invitrogen, A32787) were used. Plates were imaged by fluorescence confocal microscopy (Nikon, A1R-HD25) and analyzed using Nikon Elements GA3.
Azide-tagged proteins
2D ReNcell VM cultures were seeded in a 24-well plate containing 0.1-µm inserts (CELLTREAT, 230634) at approximately 75,000 cells per well. The culture was aged for 1 week in methionine-free media (Sigma-Aldrich, D9785-10L). The inserts were infected with HSV-1 (6.67 × 105 PFU per μl) at 1:250 for 24 hours. Six hours after infection, 50 µM L-azidohomoalanine was added. After infection, the inserts were removed, and the wells were fixed in 4% PFA for 1 hour at room temperature. After three washes of PBS, a permeabilizing solution of 0.5% Triton X-100 PBS was added for 15 minutes at room temperature. After three washes of PBS, a click reaction mix of 50 µM Alexa Fluor 647 Alkyne (Life Technologies, A32787), 100 mM copper sulfate and 1 M dithiothreitol in PBS were added to the wells for 1 hour at room temperature while being protected from light. The wells were then washed three times with PBS, and 5% BSA was added overnight. The next morning, three PBS washes were followed with anti-p-tau (PHF1, Albert Einstein College of Medicine) antibody at 1:500 shaking overnight at 4 °C. After three PBS washes, the plate was stained with secondary anti-mouse 594 (Invitrogen, A11032) at 1:1,000 in 5% BSA. Three more PBS washes were completed, and the wells were stored in PBS. Wells were imaged by confocal microscopy (Nikon, A1R-HD25) and analyzed by Nikon Elements GA3.
Thioflavin S co-localization
3D ReNcell VM cultures seeded in a 96-well plate (Greiner Bio-One, 655866) were aged to 3.5 weeks. At 3.5 weeks, the media were completely removed and replaced with media containing HSV-1 (2.465 × 107 PFU per well) and incubated for 24 hours at 37 °C. The wells were then washed once with PBS and then fixed with 4% PFA PBS at 4 °C for 2 hours. Afterwards, the plate was PBS washed three times followed by blocking with Doo Block at 4 °C shaking overnight. Then, anti-p-tau (PHF1, Albert Einstein College of Medicine) at 1:500 in Doo Block was added, and the plate was incubated at 4 °C shaking overnight. After overnight incubation, the plates were washed five times at 5- minute intervals using TBS-Tween. Fluorescent secondary anti-mouse 647 (Invitrogen, A32787) was added at 1:1,000 in Doo Block, and the plate was incubated on a shaker for 3 hours at 4 °C. Five 5-minute washes were repeated, and then 0.05% thioflavin S dissolved in 50% ethanol/water was added to the wells for 10 minutes. This was followed by three 80% ethanol/water washes and seven PBS washes for 10-minute intervals. After the final wash, the plates were stored in PBS at 4 °C. Plates were imaged by fluorescence confocal microscopy (Nikon, A1R-HD25), and images were analyzed using Nikon Elements GA3.
Cytoxicity assay
Three and one-half to four-week-old 3D ReNcell VM cultures were infected with serial dilutions of HSV-1 for 24 hours. Conditioned media were collected and stored at −20 °C in LDH storage buffer. The samples were later analyzed using an LDH-Glo Cytotoxicity Assay (Promega, J2381), and luminescence values were recorded from a plate reader (BioTek Synergy Neo2).
Statistics and reproducibility
Statistical parameters, tests used, sample sizes and number of repeats are noted in the figure legends. No statistical methods were used to predetermine sample sizes; sample sizes were chosen based on previous similar studies10,11. Data collection was randomized. Data collection and analysis were performed blinded to the conditions of the experiments whenever possible—however, not for all experiments. Exclusion of data points was performed based on standard GraphPad Prism outlier analysis (ROUT method, Q = 1%). Images are representative of all experimental repeats. Statistical significance between two groups was determined using two-tailed Student’s t-tests, or, in cases where assumptions of normal distribution or variance are violated, Mann−Whitney test or Welch’s t-test was used, respectively. Statistical significance for more than two groups was determined with multiple comparison ANOVAs. In cases where normal distribution assumptions were violated, a normality test was performed, and the subsequent significance test was determined accordingly. Comparisons between treatment groups only to a single control group were corrected using Dunnett’s correction. Comparisons between all groups were corrected using Tukey’s correction. In several instances, multiple independent comparisons were corrected using Sidak’s correction. Linear regression analyses were used to determine a rate of constancy between two quantitative variables. In several instances, Kolmogorov−Smirnov test was used to determine cumulative distribution function between two independent samples. All images from in vitro samples are representative of multiple image fields described in each figure legend from a minimum of three independent experiments. The threshold for determining statistical significance was P < 0.05. All statistical analyses were performed with GraphPad Prism 10.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.