ScFv-MC1 design and sub-cloning into AAV1
The light and heavy-chain variable domains corresponding to the MC1 antibody were sequenced employing the MCLAB antibody service (San Francisco, CA). As previously published [43] the VH and VL chains were joined together by a 15 amino acid residues linker (Gly4Ser)3. 5′-terminal signal peptide (SP) and 3′-terminal Myc and His6X tags were added. The AAV packaging and purification service was provided by Vector Biolab (Malvern, PA). ScFv-MC1 was sub-cloned into the adeno-associated viral vector serotype 1 (AAV1) under the control of the synthetic strong CAG (CMV-chicken beta actin-rabbit beta globin) promoter. In order to enhance expression of the transgene, the WPRE Woodchuck hepatitis virus (WPRE) post-transcriptional regulatory element was added 5′ of the Myc and His6X tags.
Tau transgenic mice
JNPL3 mice obtained from Taconic (Germantown, NY) express 0N4R human tau with the P301L mutation that causes frontotemporal dementia in humans, under the mouse prion promoter. JNPL3 mice develop NFTs-like pathology as early as 4.5 months and in later stages progressive deterioration of the motor function [62]. Homozygous P301S were obtained from Dr. Michel Goedert (Cambridge, UK) [63]: these mice, on pure C57BL/6 background, express 0N4R human tau carrying the P301S mutation, under the control of the neuron-specific murine Thy-1 promoter, and they develop widespread tau pathology affecting cerebral cortex, hippocampus and brain stem as early as 6 months, and partial paralysis of the lower limbs by 8 months of age. Animals were treated according to the current regulations for the proper handling of research animals, following an approved IACUC protocol.
ScFvMC1 purification
ScFvMC1 purification was performed as previously published [43]. Briefly, scFv-MC1 was cloned into the mammalian expression vector pcDNA3.1 (Genewiz, South Plainfield, NJ) and transfected into HEK293T, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h of transfection, the scFv released into the conditioned medium was affinity purified using a Ni-Sepharose High Performance column (GE Healthcare, Port Washington, NY). The efficiency of purification was tested using an immunosorbent assay employed to assess the antigen–binding specificity of the scFvMC1, as previously described [43]. Starting material, flow through and eluted fractions were tested to check for proper enrichment of the purified material. The purified scFv-MC1 was checked on Coomassie-stained SDS-PAGE gel for proper molecular weight.
Infrared conjugation and intravenous (IV) injections
ScFv-MC1 and MC1 have been conjugated with IRDye 800CW, using IRDye 800CW protein Labeling Kit Low-MW or High-MW respectively (LI-COR Biosciences, Lincoln, NE), according to the manufacturer instructions. Briefly, scFv-MC1 and MC1 were dialyzed in 50 mM potassium phosphate buffer pH 8.5 at 4 °C, overnight; the pH was then adjusted with 1 M potassium phosphate buffer to 9. After 2 h incubation, the unconjugated dye was removed using desalting spin columns (Zeba Desalt Spin Columns, Thermo Scientific). ScFvMC1 and MC1 antigen-binding reactivities were measured against an MC1 specific peptide [43] by immunosorbent assay, in order to exclude loss of activity upon conjugation.
To verify the ability of scFvMC1 and MC1 to cross the blood brain barrier, IV injection was performed in 3-month-old JNPL3 mice using 100 μg of the antibodies: saline, scFvMC1-IRDye, MC1-IRDye, or unlabeled antibodies were injected (n = 3 per group). Mice were anesthetized with isoflurane and the injections performed retro-orbitally. Mice were sacrificed 2 h post injection; brains were harvested and dissected into cortex (Ctx), hindbrain (HB) and hippocampus (Hip). Homogenization was performed in 1X RIPA buffer (Thermo Fisher Scientific, Waltham, MA) with the Mini protease inhibitor cocktail (Roche, Indianapolis, IN). Brain samples from each region were spotted on 0.45 μm nitrocellulose followed by IR signal acquisition at 789 nm, using Sapphire Biomolecular Imager (Azure Biosystems, Dublin, CA).
Intra-muscular (IM) injections
AAV1-CAG-scFvMC1 or AAV1-CAG-eGFP were injected at a dose of 2X1011 GC per mouse. Each AAV was diluted in PBS at a final volume of 50 μl, and a one-time intramuscular injection was administrated in the right tibialis. Injections were performed upon anesthesia with isoflurane.
Twenty-six females JNPL3 (n = 13 per group) mice were injected at 3 month of age and sacrificed 4 months later. The P301S line was injected at 2 month of age and sacrificed 4 months later; we used twelve females P301S in total (n = 6 per group). Overall, 26 JNPL3 and 12 P301S mice were employed in this study.
Brain extracts and tissues preparation
Mice were sacrificed by isoflurane overdose, decapitated and processed as described previously [14]. The brain was removed and divided at the midline so that just one half of the brain was dissected for biochemical analysis. Cortex, hippocampus and hindbrain were homogenized separately using an appropriate volume of homogenizing buffer, a solution of Tris-buffered saline (TBS), pH 7.4, containing 10 mM NaF, 1 mM Na3VO4 and 2 mM EGTA, plus the complete Mini protease inhibitor cocktail (Roche). Supernatants were analyzed for protein concentration using DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Brain homogenates were stored at − 80 °C and used for separate measurement of soluble and insoluble tau. Soluble tau was measured as heat-stable preparation (hsp) from brain. Hsp were prepared by adding 5% ß-Mercaptoethanol and 200 mM NaCl to the brain homogenates. Samples were then heated at 100 °C for 10 min and cooled at 4 °C for 30 min. After centrifuging at 14,000 g in a table-top microcentrifuge at 4 °C for 15 min, supernatants were collected and 5X sample buffer (Tris-buffered saline, pH 6.8 containing 4% SDS, 2% ß-Mercaptoethanol, 40% glycerol and 0.1% bromophenol blue) was added. To obtain insoluble tau preparations (INS), homogenates were thawed and spun at 14,000 g for 10 min at 4 °C. The collected supernatants were centrifuged at 200,000 g for 30 min at 4 °C; the pellets were then re-suspended in homogenizing buffer and centrifuged again at 200,000 g for 30 min at 4 °C. The final pellets were re-suspended in 1X sample buffer and heated at 100 °C for 10 min to efficiently dissociate the insoluble tau fraction.
Liver, kidney and heart were harvest and homogenated using 1X RIPA buffer with the complete Mini protease inhibitor cocktail (Roche). Protein concentration were analyzed using DC Protein Assay (Bio-Rad Laboratories) and samples were prepared for western blotting. For tibialis and gluteus maximum muscles protein were extracted in skeletal muscle homogenizing buffer (20 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10% glycerol, 1 mM EDTA and 1 mM dithiothreitol) plus the complete Mini protease inhibitor cocktail (Roche). Tissue were mince using a Douncehomogenizer, sonicated and then let vortexed overnight at 4 °C. The supernatant, containing the protein extract, was collected after 15 min centrifugation at 14,000 g and used in immunoblot, as described later.
Tau ELISA
Levels of total and phosphorylated tau were assessed using the Low-tau ELISA (enzyme-linked immunosorbent assay) protocol previously published [64, 65]. 96-well plates were coated for 48 h at 4 °C with specific purified monoclonal tau antibodies (DA31, CP13, PHF1, RZ3) at a concentration of 6 μg/ml. After washing, plates were blocked for 1 h at RT using StartingBlock buffer (Thermo Fisher Scientific). Brain samples and standards were diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBS and loaded on the plates. Once the samples were added, the total tau detection antibody DA9-HRP, diluted 1:50 in 20% SuperBlock in 1XTBS, was added to the samples and tapped to combine. Plates were then incubated overnight at 4 °C. Next day, 1-Step ULTRA TMB-ELISA (Thermo Fisher Scientific) was added for 30 min at RT, followed by 2 N H2SO4 to stop the reaction. Plates were read with Infinite m200 plate reader (Tecan, San Jose, CA) at 450 nm.
Immunoblotting
An aliquot of the total lysates was used for western blotting (WB). 0.1% SDS was added to the lysates, followed by sonication (3 cycles, 10 s each). Samples were run on 4–20% Criterion Tris-HCl gels (Bio-Rad Laboratories) and electrophoretically transferred to a nitrocellulose membrane (Thermo Fisher Scientific). Residual protein-binding sites were blocked by incubation with 5% non-fat milk in 1XTBST (1X TBS plus 0.1% Tween 20) 1 h at RT, followed by an overnight (O/N) incubation at 4 °C with primary antibodies diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBST. Mouse anti-tubulin (Thermo Fisher Scientific) were diluted 1:5000; anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was diluted 1:1000. Appropriate isotypes secondary antibodies HRP-conjugated were diluted 1:2000 or 1:10000 in 5% non-fat milk 1XTBST, and added for 1 h at RT. Every step was followed by 3 or 4 washes in 1X TBST. Detection was performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific) and exposed to x-ray films.
Immunocytochemistry, immunofluorescence and image analysis
Tau staining and immunofluorescence were performed according to standardized protocols [14, 43]. After decapitation, half of the brain was fixed overnight in 4% paraformaldehyde at 4 °C. Serial sections were cut from the fixed brain half on a vibratome, conserved in TBS (50 mM Tris, 150 mM NaCl, pH 7.6)/0.01% NaN3, and stained on 24-well plates with a panel of tau antibodies. Endogenous peroxidases were quenched with 3% H2O2/0.25% Triton X-100/1XTBS for 30 min. Non-specific binding was blocked with 5% non-fat milk-1XTBS for 1 h at RT. Primary antibodies were used as follows: anti tau antibodies RZ3 and MC1 (1:500), CP13 and PHF1 (1:5000); all antibodies were diluted in 5% non-fat milk-1XTBS, and incubated O/N at 4 °C, shaking. Biotin-conjugated secondary antibodies (SouthernBiotech, Birmingham, AL) directed against the specific isotypes were diluted 1:1000 in 20% SuperBlock, left for 2 h at RT, and lately Streptavidin-HRP (SouthernBiotech) was incubated for 1 h. Staining was visualized by 3,3′-Diaminobenzidine (Sigma-Aldrich, St. Louis, MO). Images were acquired using Olympus BH-2 bright field microscope (Waltham, MA); analyzed and processed using ImageJ/Fiji software (NIH). Semi-quantification was done on the hippocampal quadrant CA1 and on the entorhinal cortex by using the measure particles tool, working with 8-bit images and adjusting the threshold.
For immunofluorescence, sections were pre-incubated 5 min at RT in 1XTBS (Gibco, Carlsbad, CA) containing 0.2% TritonX100 (Sigma-Aldrich). After blocking 1 h at RT with a solution containing 5% normal goat serum (Sigma-Aldrich) diluted in 1XTBS/0.2% Triton, sections were incubated with primary antibodies diluted in 1% normal goat serum in 1XTBS/0.2% Triton: anti-Myc-Alexa Flour555 1:500 (EMD Millipore), Iba-1 1:1000 (Wako Chemicals, Richmond, VA), anti-CD68 1:200 (Bio-Rad Laboratories) and RZ3 (anti-tau pThr231) 1:500. After washing 3X in 1XTBS/0.2% TritonX100, Alexa Fluor secondary antibodies − 488 and − 568 and − 350 (Invitrogen) were added at 1:1000 or 1:2000 dilutions for 1 h at RT, in different combinations in order to obtain multiple labeling images. DAPI (Invitrogen) was used to counterstain. Brains slices were then mounted on slides and let dry 20 min before being coverslipped using Vectashield hard set anti-fade mounting (Vector Laboratories, Burlingame, CA). Sections incubated without primary antibody were used as negative controls. Images were acquired using Zeiss 880 confocal laser microscope (Peabody, MA). Integrated intensity was quantified using NIH ImageJ (NIH) on raw images, with background fluorescence subtraction on pre-defined ROIs.
For Iba-1 VIP-substrate staining (Vector Laboratories, Burlingame, CA), antigen retrieval was performed using 1X Dako Target Retrieval solution (Agilent Dako, Santa Clara, CA, USA) in distilled water/0.5% Triton, at 95–99 °C for 5 min. After washing, endogenous peroxidases were quenched with 3% H2O2/0.25% Triton X-100/1XTBS for 30 min. Sections were incubated in 5% normal goat serum (Sigma Aldrich) in 1XTBS/0.1% Triton. Primary polyclonal antibody, anti Iba-1 (Wako Chemicals, Richmond, VA), was diluted 1:2000 in 1% normal goat serum in 1XTBS/0.1% Triton and let incubate O/N at 4 °C. Biotin-conjugated goat anti-rabbit secondary antibody (SouthernBiotech) were used at 1:2000 in 20% SuperBlock (ThermoFisher) in 1X TBS/0.05% Triton, left for 2 h at RT, and lately Streptavidin-HRP (SouthernBiotech) was incubated for 1 h. Staining was visualized using Vector VIP Substrate (Vector Laboratories) following the manufacture’s specifications. After washing with distilled water slides were mounted and coverslipped. Microglia were imaged on AxioImager Z1 microscope (Zeiss) at 63x oil and 0.58 μm z-steps to capture 3 ROIs across the stratum radiatum of the CA1 subfield of the hippocampus for each animal (5 mice per group, 10 cells per mouse imaged: 50 cells per treatment group analysed). The microglia process morphology was categorized with a score from 0 to 3, following the criteria described by Schafer et al. [66,67,68]: 0 (> 15 thin processes with multiple branches), 1 (5–15 thick processes with branches), 2 (1–5 thick processes with few branches), 3 (no clear processes). All analyses were performed in blind.
On peripheral organs, histology was performed by HistoWiz Inc. (histowiz.com) using a Standard Operating Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4 μm. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with enzyme treatment (1:1000) using standard protocols. Slides were stained with hematoxylin and eosin and anti-NFkb. Bond Polymer Refine Detection (Leica Biosystems) was used according to manufacturer’s protocol. After staining, sections were dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40X) was performed on an Aperio AT2 (Leica Biosystems).
Primary mouse microglia cultures and uptake experiment
Cultures were prepared from post-natal C57BL/6 mouse pups at 2 days of age. Whole brains were trypsin digested and made into a cell suspension. Cells were seeded in flasks pre-coated with 0.1 mg/ml poly-D-lysine (Sigma-Aldrich) and maintained in DMEM supplemented with 10% heat-inactivated FBS (Gibco) and 1% Pen-Strep (Gibco). Medium was supplemented with 5 ng/ml Macrophage Colony Stimulating factor (M-CSF) (Thermo Fisher Scientific) diluted in PBS supplemented with 0.1% sterile filtered BSA (Sigma-Aldrich). At DIV10 microglia were isolated by orbital shaking at 150 RPM for 1 h and the supernatant was seeded in 12-well plates with 300,000 cells per well. Experiments were performed on the subsequent day. PHF-tau (paired helical filaments) [69] was added to microglia at a concentration of 1 μg/ml as determined by total tau ELISA. ScFvMC1 was added at a concentration of 10 μg/ml. To allow for immune complex formation, PHF-tau and scFvMC1 were mixed in medium and pre-incubated at 37 °C for 30–45 min prior to addition to cells. Mixing was performed two times during incubation by repeated manual pipetting. The 2 h incubation was performed in medium without serum. All experiments were performed in triplicate, with each treatment group in quadruplicate. The amount of PHF-tau in medium at the end of the experiments was assessed using the same low-tau ELISA previously described.
Stereotaxic intracranial injection
Intra-hippocampal injections of AAV vectors were performed according to a stereotaxic surgery protocol previously published [43]. Briefly, under sterile conditions, 3-month-old P301S mice were anesthetized and secured on a stereotaxic frame (David Kopf instruments, Tujunga, CA). Mice received bilateral hippocampal injection of AAV preparations using a neuro syringe with a 33 gauge needle (Hamilton, Reno, NV), using the following coordinates: AP − 2.1 from bregma, ML +/− 2.0 from bregma, DV − 1.8 below dura. Animals were treated according to the current regulations for the proper handling of research animals, following an approved IACUC protocol.
Flow cytometry on adult mice microglia
Microglia was isolated from 6-month-old P301S mice treated with AAV5-scFv-MC1 and AAV5-null injected mice. In this experiment, we used an AAV-null construct instead of AAV-eGFP, since our goal was to ascertain the uptake of the scFv by microglia and since eGFP may interfere with flow cytometry analysis. Mice were anesthetized and cold PBS-perfused. After dissection, the forebrain was minced with a Dounce homogenizer in ice cold HBSS, filtered onto 70 μm cell strainer, and centrifuged at 300 g for 5 min at 4 °C. Tissue was dissociated using Neural Dissociation Kit P (MACS Miltenyi Biotec, Auburn CA) according to the manufacturer’s instruction. Myelin debris were removed using Myelin Removal Beads II (MACS Miltenyi Biotec). Briefly, after neural dissociation, samples were spun at 300 g for 10 min at 4 °C and incubated 15 min with Myelin Removal Beads in 0.5% BSA in 1X PBS. Cells suspension was then loaded onto a pre-washed MACS LS column and placed in the magnetic field of MACS Separator. The magnetically labeled myelin was retained within the column while unlabeled cells run through [70]. Cells suspension, myelin depleted, was then washed twice with FACS buffer (0.05% BSA, 0.02% sodium azide in 1X PBS) and stained with Live/Dead-Pacific Blue (Thermo Fisher Scientific). Surface staining was performed using CD11b-PE and CD45-APC/Cy7 antibodies (BD Biosciences, Franklin Lakes, NJ) in order to select microglia from other monocytes. After fixation and permeabilization with BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) cells were stained with anti-Myc Tag Antibody AlexaFluor-647 (Thermo Fisher Scientific). Debris, doublets and dead cells were excluded using fsc/ssc, fsc-h/fsc-w and Pacific Blue gates, respectively. BD CompBeads (BD Biosciences) were used for calibration of flow cytometer. Samples were analyzed on a BD LSRFortessa and data processed using FlowJo software (Treestar).
Tau and anti-scFvMC1 antibodies detection in serum
A detailed protocol was previously published to detect total tau in serum [71]. Upon sacrifice mice were bled, samples collected and allowed to clot for 30 min at RT. After cooling for 15 min, samples were spun at 14,000 g for 10 min at 4 °C; supernatants were collected and then re-spun at 14,000 g for 5 min at 4 °C. The final supernatants correspond to the serum samples. In order to detect tau in serum, samples were diluted 1:3 in 0.2 M NaOAc, pH 5.0 and heated at 90 °C for 15 min. After the heat treatment, samples were allowed to cool at 4 °C for 15 min, and then spun at 15,000 g for 10 min. Supernatants were collected and 1 M Tris buffer was added to neutralize the pH. After diluting 1:2 in 20% Superblock, samples were loaded on the total tau ELISA (DA31 capture).
In order to detect antibodies directed to the scFvMC1, 96-well plates were coated with purified scFvMC1 at 6 μg/ml for at least 24 h. Plates were washed 3X and blocked for 1 h using StartingBlock (Thermo Fisher Scientific). Plates were washed 5X and 50 μl of sera added in triplicate at 1:1000 dilution in 20% SuperBlock (Thermo Fisher Scientific). After 1 h incubation plates were washed 5X and 50 μl of goat anti-mouse non-specific IgG HRP-conjugated (SoutherBiotech, Birmingha, AL) was added and incubated for 1 h. Finally, Bio-Rad HRP Substrate Kit has been used for the detection and plates were read with Infinite m200 plate reader (Tecan) at 415 nm.
Statistical analysis
Quantitative data were analyzed using the dedicated software GraphPad Prism V.6 (GraphPad software Inc., CA). Unpaired t test with Welch’s correction was performed when the parametric assumption of normality (D’Agostino-Pearson omnibus test) was accomplished. When not, non parametric Mann-Whitney test was performed instead. Statistical significance was set at P < 0.05. Error bars represent the standard error of the mean (SEM).