Mice

Tau−/− mice were generated by knockin of green fluorescence protein (GFP) encoding cDNA into the first exon of the endogenous Mapt locus as described before68 (available from JaxMice #004779). Mice were maintained on a C57Bl/6 background. Three to 6 months old male mice were used throughout the study at indicated N-numbers. Experimenters were blinded to the randomly assigned genotype or type of AAV injected for all experiments until after analysis was completed. Blinding and sample/animal randomization was done by staff not involved in the study. All procedures were approved by the Animal Ethics Committee of the University of Sydney and the University of New South Wales, Australia.

Middle cerebral artery occlusion

Transient MCAO was used to induce stroke in adult mice69. Accordingly, male C57Bl/6 mice (age: 3–6 months; body weight: 25–35 g) were anesthetized and placed on their backs to expose the neck area. The common (CCA), external (ECA) and internal carotid arteries (ICA) were exposed via a ventral midline neck incision and connecting tissue around the vasculature was removed. The distal ECA was tied off and opened by arteriotomy and a heat-blunted 5-0 nylon monofilament was inserted and gently advanced upwards, ~10 mm past the CCA bifurcation. Reduction in cerebral blood flow, as determined by transcranial laser Doppler flowmetry (Moor instruments), confirmed MCAO. The monofilament was withdrawn after 1.5 h or 30 min (for long-term follow up) under continued anesthesia. Body temperature was maintained and monitored by placing mice on rectal probe-controlled heat pads (Kent Scientific Corporation) for the duration of the entire procedures. Mice were individually housed after full recovery from anesthesia. Neurological severity scoring (NSS) was done at indicated time points, according to the following (Supplementary Fig. 1): grade 0, no deficits; 1, decreased resistance to lateral push; 2, limb extension; 3, limb elevation; 4, circling. Animals were terminated at indicated time points after onset of MCAO. Mice for long-term follow up (30 min MCAO) were weighed and neurologically scored daily. In sham operated controls, the carotid arteries where exposed and the ECA cauterized before the skin was closed again. Mice that had bleeding complications during the surgery or when the filament was removed were excluded from the study and not counted to the total numbers examined. This occurred in less than 10% of mice, and as frequently in tau−/− and tau+/+ mice. Blood pressure was recorded from the tail with CODA Surgical Monitor. Heart rate, peripheral O 2 saturation and body temperature were monitored using a PhysioSuite system (both Kent Scientific Corporation). Blood gases and electrolytes from whole blood were analyzed with an i-STAT Handheld device and CG8 + cartridges (Abbott).

Rota-rod testing

Rota-Rod testing of mice that have undergone 30 min of transient MCAO was performed as previously described70. Mice were trained on the accelerating-mode Rota-Rod (4–40 r.p.m. over 4 min) for 5 consecutive days prior to the surgery. Testing was done every 2 days after MCAO.

Cortical NMDA infusion

Stereotaxic surgeries were done as previously described71. Briefly, mice were anaesthetized with isoflurane and mounted in a stereotaxic frame (KOPF). The skin over the skull was opened and the bone exposed and cleaned. A small burr hole to allow injection into the brain was drilled into the skull. Coordinates for cortical injections were: AP: −2 mm, RL: 1.2 mm, DV: 1 mm. NMDA (50 mM; 0.2 µl) was infused over 1 minute into the cortex of mice under isoflurane anesthesia. The needle was left in place for another 5 min. After the surgery, mice were removed from the frame and the skin closed with sutures. All surgeries were done under aseptic conditions. Mice were transcardially perfused 24 h after the surgery, brains removed, paraffin embedded and serial sections stained with a standard Nissl protocol. The area damaged was determined on 10 serial 10 µm sections at 100 µm intervals.

Adeno-associated virus vectors

A KpnI-linkered DNA fragment, entailing the mouse U6 promoter and a SynGAP1 small hairpin (sh) RNA (ccagaaccctctcttccatat), was synthesized (Epochbiolabs, Missouri City, USA) and cloned into a rAAV plasmid containing the CAG promoter driving a humanized renilla GFP reporter (Adeno-associated virus (AAV)-SG1-shR). The same backbone with an EGFP shRNA replacing SynGAP1 served as a control (AAV-ctr-shR). Packaging of rAAV1 vectors was performed as described72. One µl of either AAV-SG1-shR or AAV-ctr-shR vector (2 × 1012 viral genomes/ml) was injected bilaterally into the striatum ( + 4.0mm AP, ± 1.8 mm ML, −2.3 mm DV from lambda), thalamus ( + 2.0 mm AP, ± 1.7 mm ML, −2.5 mm DV) and cerebellum (−2.3 mm AP, ± 2.0 mm ML, −2.8 mm DV) of cryo-anaesthetized neonatal mice as described73.

Electroencephalography

Methodology of electroencephalography has been previously described74. Briefly, after anesthesia with ketamine/xylazine and induction of MCAO, the recording electrode on remote telemetric transmitters (DSI) was implanted in the cornu ammonis (CA) region of the hippocampus (−2.0 mm AP, + 2.0 mm ML, −2.0 mm DV from bregma) and the reference electrode placed above the cerebellum (−6.0 mm AP, 0 mm ML, 0 mm DV). Local field potentials (LFPs) were recorded through amplifier matrices (DSI) at 500 Hz sampling rate (Dataquest A.R.T.). Raw LFPs were noise filtered using a powerline noise filter (DSI). Epileptiform discharge analysis of EEG recordings was performed using NeuroScore software v3.0 (DSI) with integrated spike detection module. Fast Fourier transform-based spectral analysis of interictal sequences was performed using NeuroScore software v3.0 (DSI). Average amplitude envelope time series were obtained by Hilbert transformation of filtered LFPs (MATLAB).

PTZ administration

To induce excitotoxicity, 6 weeks-old mice were administered pentylenetetrazole (PTZ; 50 mg/kg bodyweight i.p.)21. Directly after the injection, mice were individually placed into a 40 × 40 cm box to observe the development of seizures. Seizure severity rating was undertaken by an independent, blinded person as follows: 0, no seizures; 1, immobility; 2, tail extension; 3, forelimb clonus; 4, generalized clonus; 5, bouncing seizures; 6, full body extension; 7, status epilepticus.

Primary neuronal cultures and staining

Primary neurons were obtained from 16 days-old tau+/+ and tau−/− embryos75. Briefly, the abdominal cavity of time-mated females was opened to remove the uterus. Embryos were placed on ice, decapitated and brains removed. After meninges were carefully removed, cortices and hippocampi were dissected and incubated with trypsin (Sigma) at 37 °C for 15–20 min, followed by trituration with fire-polished glass Pasteur pipettes (Livingstone) to obtain single cell solutions. Cells were counted using a hemocytometer and plated in Dulbecco’s Modified Eagle Medium (Life technologies) medium containing 10% heat-inactivated fetal bovine serum (Hyclone). Medium was changed to Neurobasal containing B27 supplement and Glutamax (all Life technologies) for continued culturing. Neurons were cultured for 15 days, and then treated with either 10 µM NMDA, 25 µM NMDA, 50 mM KCl, 5 µM forskolin or 50 µM bicuculline for 30 min before harvesting for Western blotting. To determine cell death, cells were treated with 0, 10 or 25 µM NMDA for 30 min. Then, NMDA-containing medium was removed and cells were washed twice with warm Neurobasal medium (Thermo) before conditioned medium from before treatments was added back. After a further 24 h incubation, cells were fixed with 4% PFA and mounted in Fluoromount-G (SouthernBiotech) with DAPI (Molecular Probes). Cells with condensed nuclei were considered dead76. For staining, cells were fixed at 21 days in vitro (DIV) with 4% PFA and stained with primary antibodies to SynGAP1 (Sigma) and PSD-95 (Millipore) using established protocols77. Images were taken with an Eclipse Ti confocal microscope (Nikon). DIV 4 primary neurons were transfected V5 tagged SynGAP1 (V5-SynGAP1) or mCherry control pLVX expression plasmids using Lipofectamine LTX (Invitrogen) according to the manufacturers protocol. DIV 12 neurons were pre-treated with 5 µM nifedipine, 40 µM CNQX and 1 µM tetrodotoxin citrate (all from Tocris) for 1 h, followed by treatment with 100 µM NMDA (or vehicle) for 6 min at 37 °C/5% CO 2 . Primary antibodies for staining were against phosphorylated ERK (Cell Signaling) and V5 (Sigma), and DAPI was used for nuclear visualization. Phosphorylated ERK staining intensity of randomly selected transfected neurons was quantified using ImageJ (NIH). Data from vehicle treated cells were pooled, since there was no difference between transfections.

Calcium imaging

Mice were injected at P0 with AAVs expressing the Ca2+ reporter GCaMP5G. At 1 month of age, acute brain slices (400 μm) were prepared using VT1200 vibratome (Leica) according to standard procedures. Briefly, mice were sacrificed, brains removed and sectioned coronally in modified high sucrose low sodium ice cold artificial cerebrospinal fluid (sACSF) containing 4 mM KCl, 1 mM CaCl 2 , 6 mM MgCl 2 , 25 mM NaHCO 3 , 246 mM sucrose, 10 mM glucose and the pH indicator phenol red (pH adjusted to 7.3), bubbled with carbogen (95% O 2 , 5% CO 2 ). Slices were thereafter maintained at room temperature in artificial cerebral spinal fluid (ACSF) solution containing 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl 2 , 1.5 mM MgCl 2 , 26 mM NaHCO 3, 1 mM NaH 2 HPO 4 , and 11 mM glucose, bubbled with carbogen. After equilibration of at least 60 min, slices were transferred onto a recording chamber and constantly superfused at 2 ml/min ACSF bubbled with carbogen. After recording baseline responses for 5 min, slices were exposed to 1 mM glutamate (bath applied in ACSF) for 5 min and cortical Ca2+ responses were monitored until neurons recovered back to baseline levels. Changes in GCaMP5G fluorescence in the cortex were imaged using a confocal microscope (Zeiss 710NLO LSM, 488 nm excitation; 5x/0.3 W objective). Images were taken every 10 seconds. The Zen software (Zeiss) was used to measure mean pixel intensity of the whole field.

Histology

Immunohistochemical staining and quantification of fluorescence intensity has been previously described in detail78. Briefly, paraformaldehyde fixed and paraffin embedded tissue was section on a microtome (Thermo) to 5 µm. Sections were rehydrated via xylene followed by decreasing concentrations of ethanol. For staining, sections were individually mounted in Sequenza racks (Thermo), blocked with 2% heat-inactivated goat serum (Sigma)/3% bovine serum albumin (Sigma) in PBS, before incubation with primary antibodies. Primary antibodies were visualized by incubation with Alexa-fluorophore labeled secondary antibodies (1:250, Molecular Probes) after washing with PBS. Primary antibodies were against SynGAP1 (1:100, Sigma), MAP2 (1:500, Sigma), Arc (1:100, SantaCruz), pH2AX (1:200, Chemicon), NeuN (1:500, Chemicon), hrGFP (1:250, abcam) and Tau5 (1:250, Invitrogen). DAPI (Molecular Probes) was used for nuclear counterstaining. 1mm fresh brain slices were obtained with a brain blocker (KOPF) and stained for 10 min at 37 °C with a 2% TTC/PBS (Sigma) solution until viable tissue turned bright red. Fluorescence intensity and infarct size were determined using ImageJ (NIH). Infarct sizes were adjusted for cerebral edema using the contralateral hemisphere as control.

Cerebral vasculature visualization

Cerebral vasculature staining was performed using Indian ink gelatin solution79. Briefly, deeply anesthetized mice were perfused with PBS and cold 4% PFA via left ventricular puncture followed by slow infusion of 0.5–1 ml 50% Indian ink in 5% gelatin at a rate of 1 ml per 30 s. Perfusion was stopped prior to ink returning to the right atrium to reduce cerebral venous filling. Mice were then left to chill on ice for 10 min to allow the gelatin to set prior to careful removal of the brain.

Western blotting

For Western blotting, protein extracts were separated by SDS-PAGE followed by semi-dry transfer onto 0.2 µm nitrocellulose membranes (Invitrogen)77. Membranes were blocked with 5% bovine serum albumin (Sigma) in TBS, washed with TBS containing 1% Tween-80 (Sigma) and then incubated with primary antibodies in blocking buffer. Primary antibodies were against ERK (1:1000, Sigma), SynGAP1 (1:1000, Sigma), phospho-ERK (1:500, Cell Signaling), V5 (1:5000, Invitrogen), Tau5 (1:1000, Invitrogen), pS214 (1:1000, Invitrogen), pS422 (1:2000, Invitrogen), pS396/pS404 (1:1000, PHF-1, P. Davies), RAS (1:1000, Millipore), Psd95 (1:2000, Millipore) and Gapdh (1:5000, Millipore). Blots were visualized by HRP-coupled secondary antibodies (1:5000, Sigma), with Luminata Crescendo Western HRP substrate (Millipore), and detected and quantified in a VersaDoc Model 4000 CCD camera (BioRad) or a ChemiDoc MP system (BioRad). Membranes were stripped for re-probing as previously described77. Full membranes of all Western blots presented are provided in the Supplementary Fig. 10.

RNA purification and quantitative PCR

A RNeasy Mini Kit (Qiagen) was used to extract total RNA from mouse brain tissue and primary cultured neurons, following the manufacturer’s instructions. To remove contaminating genomic DNA, an on-column DNA-digest was performed with RNase-free DNase I (Qiagen). cDNA was synthesized from 2.5 μg of total RNA with the second strand cDNA-synthesis kit (Invitrogen). mRNA levels were determined by quantitative PCR, using a Fast SYBR green reaction mix (Invitrogen) and gene-specific primer pairs as listed in Supplementary Table 3, using a Mx3000 real-time PCR cycler (Stratagene).

Transcriptome and pathway analysis

Next generation RNA sequencing (RNA-Seq) was done by BGI-Hong Kong (China) using RNA extracted from vehicle-injected tau+/+ and tau−/− and PTZ-injected tau+/+ and tau−/− mice. PTZ mice with similar seizure score were selected for this analysis. Briefly, at least 24 million, 90 bp long read pairs per sample could be aligned unambiguously to the GRCm38/mm10 version of the mouse genome using tophat 2.0380 and bowtie 2.0.0.681 and allowing for 2 read mis-matches. Differential expression analysis was performed using Cuffdiff 2.0180 and only genes with a p-value of less than 0.05 and a fold change of more than 1.5-fold were labeled as significantly differentially expressed. Genes were labeled as lack of response, if genes were significantly differentially expressed in vehicle-injected compared to PTZ-injected tau+/+ mice and lacking or having a significantly milder response in the same direction in vehicle-injected compared to PTZ-injected tau−/− mice. Functional annotation of the significant RNA-Seq genes was performed using DAVID82. KEGG pathway83 representations were used to represent the outcome of the analysis. All sequencing data have been submitted to the GEO repository and are available under accession number GSE45703.

Active ras pull-down

GTP-bound Ras was precipitated from stimulated hippocampal slices as previously described84. Briefly, 2-month-old mice were sacrificed, brains removed and transferred into CO 2 -adjusted and ice-cold sucrose cutting solution (0.2 mM CaCl 2 , 7 mM MgCl 2 , 28 mM NaHCO 3 , 11 mM glucose, 1.25 mM NaH 2 PO 4 , 2.5 mM KCl and 241 mM sucrose). The hippocampi were removed, sliced and then incubated in CO 2 -adjusted artificial cerebral spinal fluid (aCSF) (127 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 1 mM MgCl 2 , 25 mM NaHCO 3 and 25 mM glucose) for 30 min at 37 °C. Slices were stimulated at room temperature for 10 min in modified aCSF containing 62.5 mM KCl, 4 mM CaCl 2 , no MgCl 2 , 10 μM CNQX, 5 μM d-AP, and 1 μM TTX. Reactions were terminated by replacing the medium with ice-cold lysis buffer (25 mM HEPES (pH 7.5), 150 mM NaCl, 1 % nonidet P-40, 0.25 % Na+-deoxycholate, 10 % glycerol, 10 mM MgCl 2 , 1 mM EDTA and protease inhibitor (Roche)). Lysates (100 μg) were incubated with recombinant Raf-RBD coupled to beads to precipitate activate Ras.

Co-immunoprecipitation

Interaction of proteins was determined by co-immunoprecipitation experiments21. Briefly, brain tissue or cells were homogenized in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 (all Sigma) and complete proteinase inhibitor (Roche). After clearing by centrifugation, 200 µg of protein was incubated with antibodies over night at 4 °C. Antibodies used for precipitation were against SynGAP1 (1:200, Sigma), Tau1 (1:200, Millipore), Tau5 (1:200, Invitrogen), RasGRF (1:200, SantaCruz) and 4RTau (1:200, Dako). Antibodies were then captured with magnetic protein G beats (Invitrogen) and washed with lysis buffer and increasing NaCl concentrations (150-250-450 mM) before adding sample buffer for subsequent Western blotting. HEK293T cells (ATCC) were transiently transfected with Flag-PSD-95, tau and V5-SynGAP1 expression plasmids as previously described22.

Synaptosome preparations

Synaptosomes were purified from mouse brains using a differential extraction procedure21; First, tissue was homogenized on ice in a Sucrose Buffer containing 0.32 M sucrose, 1 mM NaHCO 3 , 1 mM MgCl 2 and 0.5 mM CaCl 2 . Then, homogenates were cleared by two rounds of centrifugation (1400 × g/10 min/4 °C). The supernatants from both spins were combined, cleared again by centrifugation (720 × g/10 min/4 °C), and then crude synaptosomes were pelleted by high-speed centrifugation (13,800 × g/10 min/4 °C). Pellets were resuspended in 300 µl Sucrose Buffer, layered on top of 1 ml pre-cooled 5% Ficoll and high-speed centrifuged (45,000 × g/45 min/4 °C). Supernatant were discarded, pellets resuspended in 100 µl pre-cooled 5% Ficoll, and layered on top of 1 ml pre-cooled 13% Ficoll for the next high-speed centrifugation (45,000 × g/45 min/4 °C). The resulting interface contained the purified synaptosomes, and was recovered carefully. Purified synaptosomes were extracted from the interfaces by diluting them with Sucrose Buffer followed by pelleting with high-speed centrifugation (45,000xg/45 min/4 C). Pure synaptosomes were further fractionated to obtain soluble, membranous and PSD-associated proteins; Therefore, pellets were resuspended in 40 mM Tris-HCl (pH 6) containing 2% Triton X-100, 0.5 mM CaCl 2 (all Sigma) and complete protease inhibitors (Roche), followed by incubation (15 min/4 °C) and high-speed centrifugation (40,000 × g/30 min/4 °C). The supernatants were recovered as soluble protein fraction. Pellets were washed, using the same 40 mM Tris-HCl buffer, incubation and centrifugation conditions as in the prior step. Then, the pellets were resuspended in 20 mM Tris-HCl (pH 8) containing 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% sodiumdeoxycholate, 0.1% SDS, 1% Triton X-100 (all Sigma) and complete protease inhibitors, followed by incubation (15 min/4 °C) and high-speed centrifugation (40,000 × g/30 min/4 °C). The supernatants were recovered as membranous protein fraction. Again, pellets were washed using the conditions of the prior step. The final extraction was done by resuspending the pellets in 5% SDS, sonication and high-speed centrifugation (20,000 × g/10 min/4 °C). The final supernatants resembled the PSD-associated protein fraction.

Duolink proximity ligation assay

Primary neurons were fixed at 21 DIV with 4% PFA for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were incubated in Duolink blocking solution (Olink Bioscience) for 30 min at 37 °C, followed by incubation with monoclonal mouse Tau5 (Invitrogen), rabbit SynGAP1 (Sigma) and chicken β3-tubulin (Chemicon) primary antibodies diluted in Duolink Antibody Diluent at for 1 h room temperature. The ligation assay was then conducted according to the manufacturer’s instructions (Olink Bioscience) and β3-tubulin was detected using A488-labeled anti-chicken secondary antibody (Molecular Probes). Images were taken with an Eclipse Ti confocal system (Nikon).

Statistics

Pre-study sample size calculation was based on decreased susceptibility of tau−/− mice to induced excitotoxic seizures, previously shown by us21. To detect a 40% reduction in infarct size (σ = 0.2) with a power of 0.95 and α = 0.05 we required a sample size of 8 (N = 7.61, Cohen method). Based on our tau−/− MCAO data, we calculated a pre-study sample size of 4 (N = 3.21) to detect a 75% difference in infarct size for the SynGAP1-knockdown MCAO study. Statistical analysis of results was done with the Prism 6 software (GraphPad Software, USA), with tests used indicated in figure legends. Values are given as mean ± s.e. All experiments were repeated at least three times.

Data availability

All sequencing data have been submitted to the GEO repository and are available under accession number GSE45703. All other relevant data are available from the authors upon request.