KEY RESOURCES TABLE
REAGENT / RESOURCE
|
SOURCE
|
IDENTIFIER
|
Antibodies
|
|
|
Tau-5
|
Abcam
|
ab80579
|
AT8
|
Thermo Scientific
|
MN1020
|
PHF1
|
Dr. Peter Davies
|
N/A
|
β-actin
|
Cell Signaling
|
4967S
|
Tubulin
|
Abcam
|
ab4074
|
MAP2
|
Abcam
|
ab5392
|
p-GR
|
Cell Signaling
|
4161S
|
Anti-Tau, 15-25(Tau-13)
|
BioLegend
|
835201
|
Chemicals
|
|
|
NaClO3
|
Sigma
|
403016
|
methyl-β-cyclodextrin
|
Sigma
|
C4555
|
dexamethasone
|
Sigma
|
D2915
|
mifepristone
|
Selleckchem
|
S2606
|
TDZD-8
|
Sigma
|
T8325
|
TTX
|
Sigma
|
554412
|
EGCG
|
Sigma
|
E4143
|
2-deoxyglucose
|
Sigma
|
D8375
|
Critical Assays
|
|
|
Total Tau Mouse ELISA Kit
|
Thermo Scientific
|
KMB7011
|
Tau (Phospho) pS199 Mouse ELISA Kit
|
Thermo Scientific
|
KMB7041
|
LDH Activity Assay Kit
|
Thermo Scientific
|
C20300
|
ATP Assay Kit
|
Abcam
|
Ab83355
|
ExoView R100 (Leprechaun)
|
Unchained Labs
|
|
Experimental Models: mice
|
|
|
C57BL/6
|
National Institute on Aging (NIA)
|
N/A
|
PS19
|
The Jackson Laboratory
|
#008169
|
Recombinant DNA
|
|
|
AAV.CAG.eGFP.P2A.hTau(P301L).WPRE.Rbg
|
Addgene
|
#140425
|
Software
|
|
|
Fiji/ImageJ
|
NIH
|
https://imagej.nih.gov/ij/
|
GraphPad Prism version 9
|
GraphPad Software
|
https://www.graphpad.com/scientific-software/prism/
|
ExoView R100 system
|
Unchained Labs
|
V-R1001000100064
|
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Clarissa Waites ([email protected])
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals
Male and female C57BL/6 mice (obtained from the National Institute on Aging) and PS19 mice (obtained from The Jackson Laboratory; strain #008169) between the ages of 4-5 months were maintained under standard laboratory conditions with ad libitum access to food and water. All animal studies were carried out with the approval of the Columbia Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health guidelines for animal care.
METHOD DETAILS
Primary hippocampal culture
Primary mouse hippocampal neurons were prepared from postnatal day 0 wild-type or PS19 mice, as described previously57, and maintained in 24-well plates with Neurobasal medium supplemented with B27, 600 μM L-glutamine, and antibiotic-antimycotic (all from ThermoFisher/Life Technologies). At 11-12 days in vitro (DIV), media was replaced with new media containing 0.5% B27 supplement and treated as follows: control (50% PEG400 diluted into media (vehicle for dex/mifepristone), dexamethasone (dex, 1µM) for 48 hours, mifepristone (5 µM) for 1-hour pre-treatment + 48 hours together with dex, NaClO3 (50mM) for 24-hour pre-treatment + 48 hours together with dex, methyl-β-cyclodextrin (1mM) for 24-hour pre-treatment + 48 hours with dex. For all conditions, media was collected at 14 DIV. For LDH measurements, media was collected from 14 DIV PS19 neurons with indicated treatments.
Brain slice perfusion
Brains were harvested from mice sacrificed via cervical dislocation without anesthesia followed by decapitation58. Brain slices including cortex and hippocampus (coronal sections; 400mm) were cut and maintained in an interface chamber at 29°C and perfused with artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O2 and 5% CO2. ACSF composition was as follows: 124 mM NaCl, 4.4 mM KCl, 1 mM Na2HPO4, 25 mM NaHCO3, 2 mM CaCl2, 2 mM MgCl2 and 10 mM glucose. ACSF was collected from ex vivo brain slices after the following treatments: control (50% PEG400 diluted into ACSF (dex/mifepristone vehicle)), dexamethasone (5 µM) for 4 hours, mifepristone (5 µM) for 1-hour pre-treatment + 4 hours with dex, NaClO3 (100mM), methyl-β-cyclodextrin (2.5mM), or EGCG (50µM) for 1.5-hour pre-treatment + 4 hours with dex.
Media/ACSF preparation for immunoblot and ELISA
When indicated, the cell culture media or ACSF were centrifuged for 20min at 2000g to eliminate cell debris, then concentrated using Pierce™ Protein Concentrators PES with 30K molecular-weight cutoff (Thermo Scientific, 88531). To deplete extracellular vesicle (EVs), media/ACSF was subjected to sequential centrifugation steps: 30 min at 10,000 g, 30 min at 21,000 g, and finally 70 min at 100,000 g. The remaining supernatant was used for immunoblotting and ELISA.
CSF collection
Five-month-old C57BL/6 mice (13/group; 10 male and 3 female) were administered dexamethasone (D2915, Sigma; 5mg/kg per day, dissolved in PBS, by intraperitoneal/i.p. injection), and mifepristone/RU486 (S2606, Selleckchem; 10mg/kg per day, dissolved in 50% PEG400 in PBS, by i.p.) for 15 days. Control animals received injections of 50% PEG400 diluted in PBS. Following this treatment regimen, mice were euthanized by isoflurane and CSF was collected from the cisterna magna using a glass capillary.
Chronic unpredictable stress, brain tissue collection, and media harvest
Three- to four-month-old wild-type animals (C57BL/6J) were housed in groups of 5–6 per cage under standard environmental conditions with ad libitum access to food and water. For the chronic unpredictable stress (CUS) protocol, animals were subjected to different stressors (i.e. 3 hours overcrowding, 3 hours rocking platform, 3 hours restraint, 30 min hairdryer; one stressor per day) that were chosen randomly to prevent habituation, over a period of six weeks. Following the CUS protocol, animals were euthanized, brain tissue was immediately macrodissected and incubated in EV-release medium (Neurobasal medium, 1% Glutamax, 1% Anti-anti; ThermoFisher) for 16h at 37ºC, 5% CO2. Five hemi-cortices were pooled to obtain each cortical sample while hippocampi from 5 mouse brains were pooled into each hippocampal sample. After the incubation period, media was collected and subject to extracellular vesicle depletion as described above (Media/ACSF preparation).
ExoView Imager Analysis
The characterization and quantification of exosomes in hippocampal culture media were performed according to the manufacturer’s instructions59. Briefly, chips containing capture probes coated with antibodies against two exosome-enriched tetraspanins, CD81 and CD9, were pre-scanned to acquire baseline particle adhesion prior to sample incubation. Media samples were diluted to fall within the dynamic range of the Exoview R100 instrument (Unchained Labs), and incubated overnight at room temperature on the pre-scanned chips in a sealed 24-well plate. The chips were then washed to remove any non-captured material, incubated for 1 hour at room temperature with fluorescently-conjugated antibodies against CD9, CD63, and CD81, washed again, dried, and then scanned with the ExoView R100 system to obtain data on particle counts, size, and exosome surface membrane protein profiles. For each capture probe (CD9 and CD81), background particle readout is subtracted from the final particle count to produce a final exosome count readout.
Immunoblotting
The concentrated media/ACSF with extracellular vesicle (EV) depletion were prepared in 4x Laemmli buffer and then boiled for 5 min, followed by SDS/PAGE (10% Tris-Glycine gel; XP00105BOX, Invitrogen), then transferred to a nitrocellulose membrane (10600001, Amersham). After blocking in TBST buffer (20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween-20) containing 5% (wt/vol) nonfat dry milk for 1 h at room temperature, the membrane was incubated with primary antibodies overnight at 4oC, then with secondary antibodies for 1 h at room temperature. The following antibodies were used: Tau5 (ab80579, Abcam), AT8: anti-phospho-Tau pSer202/Thr205 (MN1020, ThermoFisher Scientific), PHF-1: anti-phospho-Tau pSer396/Ser404 Tau (from Dr. Peter Davies), β-actin (4967S, cell signaling), anti-Tubulin (ab4074, Abcam). IRDye 800CW goat anti-mouse IgG secondary antibody (P/N: 926-32210, LI-COR), IRDye 680CW goat anti-rabbit IgG secondary antibody (P/N: 926-68071, LI-COR). Membranes were visualized by Odyssey Infrared Imager (model 9120, LI-COR Biosciences), and relative optical densities of bands determined by Fiji/ImageJ software.
ELISA
EV-depleted media/ACSF samples (50 µL volume) were used for measurement of Tau concentration by a mouse-specific total Tau ELISA kit (KMB7011, Thermo Scientific) or pS199 Tau ELISA kit (KMB7041) according to manufacturer’s instructions.
Tau uptake assay
Media was collected from donor WT or PS19 neurons treated with vehicle control or dex (1µM) for 48 hours. The media from these cultures was then depleted of EVs as described above and transferred to naïve recipient wild-type neurons for a 48-hour incubation. For one condition, recipient neurons were also treated with dex (1µM) during this time. Following incubation, recipient cells were washed three times with cold 1x PBS and fixed with 4% paraformaldehyde as previously described57. The uptake of hTau was then detected by immunostaining with MAP2 and Tau13 antibodies as described below.
Immunofluorescence staining of brain slices, cultured neurons.
Floating brain sections or fixed primary neurons were immunostained as previously described 57. Briefly, fixed neurons or slices cut at 35 μm on a vibratome (VT1000S; Leica) were incubated overnight with the following primary antibodies: mouse Anti-Tau, 15-25(Tau-13) antibody (1:1000, 835201, BioLegend) and chicken MAP2 (1:5000, ab5392, Abcam). They were then incubated for 1 h with secondary antibodies (Alexa Fluor® 594 anti-mouse IgG, and Alexa Fluor® 633 anti-chicken IgG, 1:2000 dilution). Coverslips were mounted with VectaShield (Vector Laboratories) and sealed with clear nail polish. Images were acquired with a 63X objective (Neofluar, NA 1.4) on a Zeiss LSM 800 confocal microscope running Zen2 software. The images were manually measured and quantified using the auto-threshold settings in Fiji/ImageJ software.
AAV injection procedure
The AAV.CBA.eGFP.2A.P301L-Tau plasmid, a gift from Bradley Hyman (Addgene plasmid #140425; http://n2t.net/addgene:140425;RRID:Addgene_140425), was packaged into AAV8 serotype by University of Pennsylvania Viral Vector Core. Prior to AAV injection, male/female mice (3-4/group) were administered dex (5 mg/kg, i.p.injection) +/- mifepristone (10 mg/kg, i.p. injection) or dex (5 mg/kg, i.p. injection) +/- EGCG (20 mg/kg, i.p. injection) for 7 days. Stereotactic AAV injections were performed under standard aseptic surgery conditions as previously described46. Briefly, mice were anaesthetized with isoflurane (2%), placed in a stereotactic frame (digital stereotaxic device, Stoelting Co.), and injected bilaterally with 2 ml of AAV in hippocampal region CA1 (at the following coordinates relative to Bregma: A/P −2.7 mm, M/L ±2 mm, D/V −1.5 mm) with a 10 μl Hamilton syringe at a rate of 0.25 μl/min by a Nano-injector system (Stoelting microsyringe pump, Stoelting Co.). The needle was kept in place for an additional 5 min. Afterwards, the skin over the injection site was sutured and mice were placed on a warming pad during their recovery from anesthesia. Mice were then administered dex with or without mifepristone or EGCG for an additional 14 days prior to euthanasia and brain harvest. Control animals received daily i.p. injections of 50% PEG400 in PBS (dex/mifepristone vehicle) or PBS (dex/EGCG vehicle).
Quantification and statistical analysis
All values were expressed as the mean ± SEM. All graphing and statistical analyses were performed using GraphPad Prism (GraphPad Prism9.Ink). Statistical details of experiments are provided in the figure legends. Statistical analyses were performed with unpaired, two-tailed t-test or one-way ANOVA, with appropriate corrections for unequal variances and multiple comparisons. A minimum of 3 independent replicates were used for all experiments. Values of p < 0.05 were considered statistically significant. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.