Animals
DSCR1 transgenic and DSCR1 KO mice were kindly provided by K. Baek at Sungkyunkwan University.
Ts65Dn was purchased from the Jackson Laboratory and Professor Mook-Jung of Seoul National University kindly gifted 5XFAD mice 33. 5XFAD of genetic background were maintained by crossing with C57BL/6J mice, and DSCR1/5XFAD hybrid mice were generated by crossbreeding DSCR1 transgenic mice (C57BL/6J background) with 5XFAD mice. Male mice were only used, and animal care and experiment were carried out under the guideline, approved by the Ulsan National Institute of Science and Technology (UNIST) with the Institutional Animal Care and Use Committee (IACUC).
BrdU administration
5-Bromo-2-deoxyuridine (BrdU) has been used twice a day at 150mg/kg body weight by intraperitoneal injection to mice with intervals of 12 hours during five days. After one day, ten days, and 21 days post- infection, mice were sacrificed and perfused with cold phosphate-buffered saline (PBS 0.1M, pH 7.4), followed by 4% paraformaldehyde (PFA) for brain harvest 34. Brain sections (4% paraformaldehyde post-fixed for 24 h) were additionally stored two more days in a cryoprotectant solution of 30% sucrose before freezing. Embedded brains in optimal cutting temperature compound were rapidly frozen and sectioned with a thickness of with the Leica Cryostat microtome.
Immunohistochemistry (IHC) and imaging
Floating samples of brain section were washed (3 x 5 minutes) in 0.1M PBS Wash Buffer. After washing, the sections were incubated with Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for 30 minutes at 80˚C in a dry oven. Section were left on ice to cool at room temperature (RT) and incubated at room temperature for 1 h with blocking buffer (5% normal horse serum, 0.3% Triton X-
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100 in 0.1M PBS. For BrdU staining, sections were transferred to filtered Tris-HCl (1 N, pH 7.4) during 30 minutes at room temperature, and wash the tissues (3 x 5 minutes) with Washing Buffer.
For primary staining, the samples were left with appropriate antibodies at 4°C and the samples were rinsed briefly in Washing Buffer, followed by Alexa Fluor conjugated secondary antibodies were implemented for 1 hour (37°C) or 2 hours at RT. Primary antibodies were incubated with appropriate dilutions: anti-Ki67 (1:500), anti-DCX (1:300), anti-NeuN (1:300), anti-BrdU (1:300), anti-SOX2 (1:100), anti-TET1 (1:100) and anti-GFP (1:300). A confocal microscope with the Zeiss 780 laser scanning has been used for imaging
Neurosphere isolation from Adult Dentate Gyrus
Adapted protocol for neurosphere culture was used in accordance with the previous studies 35, 36. Samples were collected from fine dissections of the adult hippocampus and were incubated in a dissociation medium with the enzyme mixture (Dispase 1U/ml, Sigma Aldrich and Papain 2.5 U/ml, Sigma Aldrich). Isolated cells through a 0.22 μm filter membrane were cultured using Neural Basal Medium (2% B27, 1x GlutaMAX, 1% Penicillin/Streptomycin, 20ng/ml EGF and FGF-2). Within two weeks, neurospheres were dispersed into a single-cell suspension using accutase, and seeded into one well of a 24-well tissue culture plate for quantitative analysis of primarily formed neurospheres.
Then, primary neurospheres were harvested and resuspended, and replated as single cells for secondary neurosphere formation assays.
The number and diameter of neurospheres were analyzed by the Zeiss Zen Blue software. To examine the differentiation capacity of Neurosphere cells (NSCs), the dispursed NSCs were cultured in the changed medium for differentiation during seven days and then stained by using each cell marker antibodies: anti-Olig2 (1: 500) for oligodendrocytes; anti-GFAP (1:300) for astrocyte; anti-Tuj1 (1:500) for neuron.
EdU using the Click-iT Imaging and FACS (fluorescence activated cell sorting)
6-8 weeks old of mice were intraperitoneally injected with 5-ethynyl-2′-deoxyuridine (EdU, 50mg/kg body weight, Sigma Aldrich) twice daily at 12-hour intervals for 3 days. On 1, 10, and 21 days after
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EdU injection, mice were sacrificed for the next experiment. 5 Hippocampi from isolated brains were dissected and then resuspended with a digestion buffer of papain and dispase. To obtain a uniform single-cell suspension, dispersed tissue cells were transiently washed with culture medium by mixing Neural Basal Medium supplemented with EGF (20 ng/ml), bFGF (20 ng/ml), 1%
Penicillin/Streptomycin, 1x GlutaMAX and 2% B27 and then sieved through a 70μm cell strainer (Corning). For Fluorescence-Activated Cell Sorting (FACS) experiment, separated cells were first fixed and permeabilized using 4% paraformaldehyde and 0.1% saponin in DPBS on the ice for 30 minutes with recombinant RNasin plus RNAse inhibitor (1:100, Promega). Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit was utilized for Edu staining, followed by primary staining with each cell type maker antibodies: anti-SOX2 (1:100), anti-DCX (1:100) and anti-NeuN (1:100) diluted into staining buffer (1% BSA in PBS-T, 1X PBS/0.5% Tween-20) for 30 minutes at room temperature. Next, Goat anti-Mouse IgG (H+L), AF647 conjugated antibody (1:300) was applied for secondary antibody staining, and then cells were sorted by FACSAria Fusion (BD Biosciences).
Immunoblotting
The cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (Roche) were added to the lysis buffer and applied to lysis tissues and cells. Protein samples were dispersed in SDS sample buffer, then separated by gel electrophoresis and transferred to PVDF. Primary antibodies were incubated with appropriate dilutions: DSCR1 (1:1000, Santacruz), TET1 (1:1000, Genetex), FLAG (1:1000, Santacruz) and GAPDH (1:1000, Santa Cruz) in the Tris-buffered saline Tween 20 (TBST) with 5% bovine serum albumin (BSA). HRP-Goat Anti-mouse or rabbit secondary antibodies (1:1000, Promega) were used.
RNA isolation and qPCR
To perform real-time quantitative PCR, the lysis of hippocampi or after FACS sorted cells was performed by TRIzol, and total RNA was extracted. The High Capacity RNA-to-cDNA Kit was used for cDNAs synthesis. LightCycler 480 II (Roche) was used according to manufacturers' instructions using PowerUp SYBR Green master mix. The 2-ΔCT method was performed as a relative quantification analysis 38. Primers used for qPCR are presented in Table 1.
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Morris water maze test and open field test
First, the MWM test was performed with the hidden platform acquisition test. All mice were trained for 5 days with 4 trials per day. And then, probe trials were conducted as described 39, 40. Briefly, mice received four trials for 1 minute with an interval of 2 minutes from different starting directions and was measured for hidden platform test. The hidden platform was removed for probe test; after training for 5 days, mice were examined for 30 seconds. To evaluate mouse movement, SMART Video Tracking software (PanLab/Harvard Apparatus) was used to record and evaluate mouse movement.
The open-field test was conducted as animals were individually placed in the center of white acrylic box (40 cm [L] × 40 cm [W] × 40 cm [H]), and behavior was tracked for 15 minutes, and the total traveled distance was evaluated by the SMART system.
Lentiviral production and stereotactic viral injection
PLL3.7 lentiviral vector that expresses Tet1 shRNA or control shRNA and CMV-EGFP reporter is included to monitor expression. HEK293T cells are co-transfected with the above lentiviral vector and a packaging vector. After 48 hours, cell supernatants were collected for virus concentration and filtered by 0.22 μm filter. Next, ultracentrifugation was performed (80,000 g, 4°C, 1.5 hours). After anesthesia with 2% isoflurane, 8-week-old mice were stereotaxically injected with the virus in the hippocampal DG using a stereotaxic apparatus. Three weeks following inoculation, mice were sacrificed.
Luciferase Assay
Firefly luciferase under different promoter constructs of miR-124 or TET1 was cloned to measure its promoter strength. For dual-luciferase reporter assays, those construction of promoter–reporter and the pRL Reporter Vectors containing control renilla luciferase, was co-expressed in Neuro2A cells. Firefly and Renilla luciferase activities were measured according to the manufacturer’s protocol using the Dual- Luciferase Reporter Assay System (Promega) 41. Then, Firefly luciferase activity was normalized with the respective Renilla luciferase activity.
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Biotinylation and biotin-streptavidin pull down assay
To produce biotin-labeled RNA in vitro, T7 RNA polymerase (Roche) was utilized to synthesize biotinylated TET1 exons and introns and U1 and U2 snRNAs with incubation of biotinylated UTP.
Biotin or no biotin-labeled RNAs were incubated with brain lysates of wild-type (WT) or DSCR1 KO mice and DSCR1 overexpressed Neuro2A cell lysates using protein lysis buffer (0.1% SDS, 0.5%
Sodium deoxycholate, 1% IGEPAL CA-630, 150 mM NaCl, 50 mM Tris-HCl pH 7.4) for 30 minutes at RT. The mixtures were furtherly incubated with pre-cleared with Streptavidin-agarose Resin (200 rpm on a rotary shaker) 42. Next, washing 3 times with ice-cold lysis buffer and elution of the resin- bound proteins were conducted, and then performed immunoblotting to detect the binding of DSCR1.
RNA stability measurement
Actinomycin D (Sigma, 5 μg/ml) was added in Neuro2A cells containing overexpression or reduction of DSCR1 in order to examine the stability of mRNA transcripts of Tet1 and decay was determined by qPCR of TET1 mRNA transcripts for the 15 hours.
Bisulfite sequencing
Genomic DNA extraction and bisulfite conversion were conducted by treating with EZ DNA Methylation-Direct Kit (TaKaRa). Amplified PCR products with specific primers from bisulfite-treated genomic DNA were purified and cloned into pMD18-T. 6 clones were randomly selected from each subject for further sequencing. The miR-124 promoter region was separated into four parts for bisulfite sequencing, and primer sequences are shown in Table 2.
Meninges isolation and immunohistochemistry
All mice were properly anesthetized using an intraperitoneal injection of the Tribromoethanol ('Avertin'), following transcardial perfusion with chilled 0.1M PBS. The skin of head was excluded by cutting the rostral tip of the nasal bone, and then the facial muscles attaching to the skull are detached. The skull
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was divided into dorsal and ventral parts at the mandibular arch, and the brain was carefully isolated from the dorsal skull. The whole-mount meninges was post-fixed with 4% PFA for 24 h at 4 °C, then detached from the skull. Isolated meninges were permeabilized and blocked in PBS with 0.05% Tween 20 (pH 6.0), 0.1% Triton X-100 containing 1% BSA, 2% horse serum for 1 h at RT. Next, primary antibodies were stored with appropriate dilutions: GFP (1:200, Abcam), Lyve-1 (1:200, eBioscience, clone), Prox-1 (1:200, AngioBio), CD31 (1:200, Millipore) in in 1% BSA in PBS-T (PBS + 0.5% Triton X-100) for 24 h at 4°C. After the tissues were washed (3 x 10 minutes) in PBST, secondary antibody incubation was implemented with alexa Fluor 488,568 or 647 conjugated goat or donkey anti-Goat, - Mouse, -Rabbi, -Rat IgG (1:500 dillution) in PBST for at least 120 min at room temperature. Samples were transferred to the DAPI solution (1:5000) for 10 minutes and were washed (3 x 10 minutes) in PBST and were mounted on slides using mounting agent (Aqua-Poly/Mount) and store in the dark at 4 °C for a subsequent microscope analysis.
Amyloid-beta staining
Hemispheres of brains were cryosectioned (40 μm) for immunofluorescence staining of Aβ. Tissues were stored in 88% formic acid (20 min, room temperature) and, after washed (3 x 10 minutes) in 1X PBS-T, the antigen retrieval by heating-induced was performed with Sodium Citrate Buffer of heating at 94°C during a half-hour. Next, samples were washed (3 x 10 minutes) in 1X PBS-T and incubated with PBS containing 0.3% Triton X-100 + 5% Horse serum for at least 1 hour at RT 43. Brain section samples were subsequently stored in adequate primary antibodies diluted with PBS (2% Horse serum, 0.3% Triton X-100): MOAB-2 (1:500, eBioscience, clone) at 4°C overnight. After rinse (3 x 10 minutes) in PBS-T, secondary antibody incubation was implemented with alexa Fluor 568 conjugated goat anti- mouse IgG (1:500 dillution) at RT for 2 hours. And then, subsequent DAPI staining (1:5000) and mounting were conducted with an Aqua-mounting solution.
Parenchyma injection
Mice were anesthetized by an intraperitoneal injection of Avertin and then head was fixed in the stereotaxis device. After the head skin was incised to expose the skull, holes were carefully drilled in the skull at the anterior-posterior (AP) axis 1.5 mm, medial-lateral (ML) axis -1.5 mm relative to
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bregma 44. Injection of 1 μl of Ovalbumin Alexa Fluor 488 conjugate was stereotaxically conducted at 2.5 mm below the skull surface with a microsyringe (Hamilton) of an injection speed (0.2 μl per minutes during 5 min) 45, and the needle was retained to avoid occurring a backflow of the injected solution at least for 5 minutes. The surgical sutures tightened the incision site, and each mouse was allowed to recover for the indicated 60 min or 120min on a heating pad. Following injection of the fluorescent tracers, brains and lymph nodes were obtained, and images were acquired on a ZEISS Axio Zoom.V16 Fluorescence Stereo Zoom Microscope equipped with an Andor's iXon EMCCD camera. Defining of OVA-AF488 efficiency is calculated by the intensity x surface area using Image J software.
Intra-cisterna magna (ICM) injection
Injection of anesthetics is typically administered by intraperitoneally with Avertin. After a midline skin incision is made, the muscles around the neck were dissected 46. Then, the cisterna magna (CM) is the surgically opened, mice were injected with 3 μL of AF 647-conjugated Ovalbumin (0.5 mg/ml) into the CM, the space filled with cerebrospinal fluid (CSF), with the Hamilton microsyringe of an injection speed (1 μL per minute), and the needle was kept for five minutes to prevent leaking of CSF15. The surgical suture tightened the muscle layer and skin. 30 minutes after the injection, mice were placed on a heating pad, and then lymph nodes were obtained for imaging.
Ligation surgery
Avertin anesthesia is used for surgery in the 5XFAD/DSCR1 TG mice at 4 months-old age. The incisional area was shaved around the neck and chest, and skin antiseptics were undergone using an antiseptic agent (70% Ethyl Alcohol and iodine). The vertical skin cut was performed in the midline from the neck to the sternum's manubrium, and then the opening was conducted by exposing the trachea, posterior belly of the digastric (PBD) muscle, sternocleidomastoid muscle (SCM) to find out the cervical lymphatic vessels (cLVs) 15. The afferent part of cLVs to the dcLN was carefully ligated using non-absorbable 10-0 nylon sutures, and sham surgery (only skin incision and exposure of lymphatic vessels) was performed. The opening site was sewed after the operation, and mice were kept warm on the heating pad for recovery. To improve the survival, mice received twice-daily subcutaneous injections of enrofloxacin (5 mg/kg) and ketoprofen (10 mg/kg) for 3 days.
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AAV virus production
For recombinant adeno-associated virus (AAV) production, the triple transfection method was used with lipofectamine (Thermo Fisher Scientific). HEK 293 cells were co-transfected with a ratio of vectors at 1:1:1 (pAAV1-CMV GFP-control vec or GFP-DSCR1 vec: pRC1 Vec: pHelper Vec) (Takara). 3 days after transfection, AAV particles producer cells were harvested, and purification of AAV particles were achieved by the AAVpro Purification Kit (Takara). The titer of viruses was determined by Real-Time PCR using AAVpro Titration Kit.
Intracerebroventricular (i.c.v.) injection
For the intracerebroventricular (i.c.v.) injection, titrated viruses were stereotaxically delivered into the left side of the lateral ventricular CSF in 4 months old AD mice. Injection sites were AP: 0.3 mm; ML:
-1.0 mm from bregma; DV: 3.0 mm from the skull via a Hamilton microsyringe with a microsyringe pump. After injection of the purified AAV1 particles (1ul, 3×1011 genome copies (GC)/ml) with an injection speed (0.2 μl per minutes during 5 min), the needle was maintained in the same site for 5 more minutes to avoid backflow. Morris water maze and Open field tests were employed 1 month after injection.
Image analysis
Using the ZEN software, images are acquired from immunofluorescent staining samples through an LSM 780 microscope. Images of maximum intensity projections were applied for quantification, and quantitive analysis was assessed using the ImageJ software. Analysis of the branch points was implemented via the Skeleton plugin of ImageJ software by counting the number of junctions from branches in the ramified network of meningeal lymphatic vessels. Lyve-1+ and CD31+ areas were measured in an indicated region of interest (ROI) of 10x confocal Z stack (5x5 tiling) images, and the number of Prox1+ lymphatic cells were also analyzed by ImageJ. Light-sheet fluorescence microscopy (Lightsheet Z.1) was used to image whole-mount meninges, and a 3D image was subsequently reconstructed using Imaris software (Bitplane) for quantitative analysis of the lymphatic volume.
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The Lyve1 positive surface ROI was additionally created to calculate of the meningeal lymphatics volume.
Magnetic-activated cell sorting (MACS)
Meninges tissues were enzymatically digested in EGM-2MV medium at 37°C for 15 minutes using 10 mg/ml of collagenase (Sigma). After filtering the tissues through a cell strainer (70 μm), singly separated cells were spun down in a cold centrifuge at 300xg for 10 min. According to the manufacturer's specifications, anti-Phycoerythrin (PE) MicroBeads were used to purify Podoplanin+ LECs with MACS. Briefly, cells were suspended with 100 μl of cold MACS buffer (0.5% BSA + 2 mM EDTA in PBS), followed by incubation with PE-podoplanin (PDPN) antibody (10 μl) for 10 minutes at 4°C. After washing with chilled MACS buffer, cells were centrifuged (300g for 10 min). Then, the pellet was suspended by pipetting up and down with 90 μL of cold buffer and stored with anti-PE Microbeads (10 μl). Cells were washed with 1ml of chilled buffer and centrifuged at 300g for 10 min.
After that, the centrifuged cells were passed through a column (LS MACS) positioned in the strong magnet of the MACS Separator. A negative fraction (Podoplanin- cells) that flowed without being filtered through the column was also collected. After washing the column 3 times with 3 ml of iced buffer, the magnetically labeled cells (Podoplanin+ cells) were directly pushed out into the tube containing chilled MACS buffer by detaching the column from the MACS Separator.
RNA extraction and RNA sequencing
According to manufacturer instructions, RNA was extracted from sorted mLECs by MACS using the Picopure RNA isolation kit. 3~5 meninges were assessed for each genotype for RNA sequencing. RNA quality was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands) with the RNA 6000 Nano LabChip kit, and ND-2000 Spectrophotometer (Thermo Inc., DE, USA) was used to quantify RNA 47. E-biogen Services Laboratory performed data analyses 48. Gene classification was applied using DAVID (http://david.abcc.ncifcrf.gov/)49.
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Statistical Analysis
Statistical significance was analyzed by the student’s t-test. For the comparison of multiple groups, a one-way ANOVA followed by Bonferroni post hoc test was generated using GraphPad Prism version 6.
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