Synaptic spine loss is one of the major preceding consequences of stroke damages, but its underlying molecular mechanisms remain unknown. Here, we report that a direct interaction of DAPK1 with Tau causes spine loss and subsequently neuronal death in a mouse model with stroke. We found that DAPK1 phosphorylates Tau protein at Ser262 (pS262) in cortical neurons of stroke mice. Either genetic deletion of DAPK1 kinase domain (KD) in mice (DAPK1-KD−/−) or blocking DAPK1-Tau interaction by systematic application of a membrane permeable peptide protects spine damages and improves neurological functions against stroke insults. Thus, disruption of DAPK1-Tau interaction is a promising strategy in clinical management of stroke.
Stroke is caused by acute brain artery bursting or cerebral embolism, leading to neuronal death and sever dysfunction of synaptic transmission (Tu et al. 2010). Loss of synaptic spine has been considered the earliest event of cerebral ischemia and generally assumed to be associated with the subsequent brain damages (Hofmeijer and van Putten 2012). Thus, protection against synaptic loss is widely considered a promising strategy in stroke therapy.
Death-associated protein kinase 1 (DAPK1), a Ca2+/CaM-dependent serine/threonine kinase, is originally identified by a functional cloning based on its involvement in interferon-γ-induced apoptosis (Bialik and Kimchi 2006). Our previous work demonstrated that following stroke, DAPK1 in the postsynaptic domain phosphorylated NMDA receptor NR2B at Serine-1303, eventually leading to neuronal death (Tu et al. 2010). Recently, we found that DAPK1 could also phosphorylate p53 at Serine-23 via direct binding of the DAPK1 death domain (DD) to the DNA-binding motif of p53 (p53DM) (Pei et al. 2014; Wang et al. 2014). Though a handful of DAPK1 substrates have been identified, downstream signaling of DAPK1 that specifically mediates early spine damage after ischemic injuries is yet to be characterized.
Tau protein colocalizes and co-immunoprecipitates with PSD95, a postsynaptic protein, and regulates the association of PSD-95 with NMDAR subunits, indicating the existence of endogenous Tau in dendritic spines probably participating in synaptic functions (Ittner et al. 2010). In addition, Tau abundantly contains high content of serine/threonine residuals and plays a crucial role in neurodegenerative diseases. There are 79 potential serine and threonine phosphorylation sites on the longest human Tau isoform. And phosphorylation of >30 of the phosphorylation sites has been reported (Morris et al. 2011). It is known that phosphorylation of these sites plays important roles in ischemic stroke-induced neuronal death (Lee et al. 2001; Zheng et al. 2010). However, the relative importance of individual phosphorylation site is still controversial.
In the present study, we demonstrated that DAPK1 directly phosphorylated Tau on Ser262, causing spine damage in the cortical neurons following stroke. We identified a binding domain of DAPK1 in the Tau microtubule repeat domains (R) with a sequence as XXIGSTENLKXX. We synthesized a membrane-permeable blocking peptide (TAT-R1D) to disrupt DAPK1-Tau binding and Tau phosphorylation. We found that systematic administration of TAT-R1D, even 6 h after stroke, at a single dose of 2 mg/kg produced therapeutic effects against stroke damages.
Materials and Methods
Adult (90 ± 5 days old) male C57BL/6J mice were used and housed individually under standard conditions of temperature and humidity and a 12 h of light/dark cycle (lights on at 08:00) with free access to food and water. Adequate measures were taken to minimize pain or discomfort during surgeries. All the experiments were carried out in accordance with the Institutional Guidelines of the Animal Care and Use Committee (Huazhong University of Science and Technology).
Generation of DAPK1-KD Knockout Mice
To identify the specific impacts of DAPK1-kinase domain (DAPK1-KD) deletion in brain function, we generated a conditional mutant strain of mice with a selective deletion of DAPK1-KD in the brain (DAPK1-KD−/− mice) by crossing DAPK1-KDloxp/loxp transgenic mice (Tg1) and CaMK2α-creERT2 mice (Tg2). For details, see the Supplementary Material.
Focal Cerebral Ischemia Model
Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO). Briefly, a 7/0 surgical nylon monofilament with rounded tip was introduced into the left internal carotid through the external carotid stump and advanced 10–13 mm past the carotid bifurcation. Occlusion was confirmed when blood flow was reduced by at least 80% of the baseline. The filament was left in place for 60 min and then withdrawn. The sham-operated animals were treated identically, except that the middle cerebral artery was not occluded after the neck incision. Each mouse was anesthetized with 2% isoflurane and maintained with 1% isoflurane in an oxygen/air mixture by using a gas anesthesia mask in a stereotaxic frame (Stoelting). The rectal temperature was maintained during surgery at 37 ± 0.5°C with a homeothermic blanket (Harvard Apparatus).
At indicated time point after ischemic reperfusion, brain was removed rapidly and frozen at −20°C for 5 min. Coronal slices (7 slices from each mouse) were made at 1 mm from the frontal tips, and sections were immersed in 2% TTC at 37°C for 20 min. The presence or absence of infarctions was determined by examining TTC-stained sections for the areas that did not stain with TTC.
AAV-eGFP Stereotaxic Injection and Dendritic Spine Analysis
Stereotactic injection of 2 µL of viral suspension containing 10E8 t.u. was at coordinates 1.94 mm posterior, 1.4 mm lateral and 2.2 mm ventral relative to bregma (Paxinos and Franklin, 2012). At indicated times after injection, the mice were anesthetized using pentobarbital (Nembutal) and perfused transcardially using 50 mL of ice-cold saline for 5 min. Brain were removed rapidly and fixed overnight in 4% paraformaldehyde for subsequent spine analysis on 40 µm free-floating coronal vibratome (VT1000S; Leica) sections. Immunofluorescent microscopy (LSM710, Zeiss) images were taken of cortical pyramidal neurons (73 cells with a total of 60 cm length of dendrites per group were analyzed) under 100 × oil lens. Images were coded and synaptic spines counted in software with Image Probes. All the spines were measured for spine densities analysis and expressed as spine/µm dendrite.
At indicated time points after ischemia, animals were deeply anesthetized and transcardially perfused with 0.9% saline followed by ice-cold 4% paraformaldehyde in 0.1 m phosphate-buffered saline (PBS); the sections were dried overnight. A TUNEL staining kit (DeadEnd™ Fluorometric TUNEL system, Promega) was used to visualize cell death in 10-μm coronal frozen sections, as per kit instructions. Labeled sections were visualized with a confocal laser-scanning microscope (LSM710, Zeiss). All labeled cells within the reticule field (0.05 mm2 at the magnification used) were counted using Image J throughout the depth of the section for 4 adjacent fields (beginning at a random starting position) of each section.
Double Immunofluorescent Staining
Animals were deeply anesthetized and transcardially perfused with saline, followed by 4% paraformaldehyde (in 0.01 m PBS, pH 7.4). Brain tissues were removed, post-fixed in 4% paraformaldehyde overnight, and then treated with 30% sucrose (in 0.1 m PB, pH 7.4) overnight till the tissue sink to the bottom. Cryosections (30 μm) were cut and stored at −20°C. Mounted brain sections were allowed to thaw at room temperature. Donkey or goat serum was used for blocking for 1 h. Next, the antibody to DAPK1 (rabbit, Cell Signaling Technology, #3008, 1:1000) and Tau (mouse,1:1000, MAB361, Millipore) or pS262 (mouse, 1:1000, SAB11111, Signalway Antibody) were incubated for 48 h. Fluorescein isothiocyanate (FITC)- and TRITC-conjugated second antibodies were then added and incubated for 1 h. The above-mentioned procedures were also used into primary cultured neurons (DIV 10) to identify the co-localizations of DAPK1 with Tau or with pS262. To visualize uptake of the peptide (Tat-R1D (IGSTENLK)-FITC) in the brain, standard immunostaining procedures were performed on free-floating frozen sections (30 μm) with overnight incubation at 4°C with chicken anti-GFAP antibody (1:1000, Abcam,ab7260) or mouse anti-NeuN (1:1000, MAB377, Millipore) or mouse anti-Iba-1 antibody (1:1000, Abcam, ab5076), and then 2 h of incubation with anti-chicken Alexa594 or anti-mouse Alexa 594 antibodies, as well as 5 min of exposure to 4′,6-diamidin-2-phenylindol (DAPI, 1:10 000) as a counterstain. Slices were mounted on gelatin-coated glass slides, cover-slipped. The sections were examined with a laser-scanning confocal microscope (LSM 510, Carl Zeiss) using an omnichrome air-cooled helium/neon laser tuned to produce beams at 488 and 594 nm. Five randomly selected sections from the whole brain sections were used. All immunoreactive positive profiles in a section were outlined, creating an artificial overlay. Measuring the average optical density of double-labeled cells was conducted on confocal images randomly taken from 2 view fields from each section, and the measuring was performed using NIH Image J software.
Co-IP and GST Affinity Binding Assays
Ischemic core of the cortex samples were dissected in cold artificial cerebral spinal fluids (aCSF) and homogenized in 200 µl 0.32 m sucrose buffer (mM) (10 sucrose, 10 HEPES, pH 7.4) containing a “complete” protease inhibitor cocktail (Roche diagnostics). Samples were centrifuged (1000 × g, 10 min, 4°C) to yield the nuclear enriched pellet and the S1 fraction. The S1 fraction was centrifuged (12 000 × g, 20 min, 4°C) to obtain supernatant (S2; microsomes and cytosol) and pellet (P2; crude synaptosomal membranes) fractions. The S2 fraction was centrifuged (160 000 × g, 2 h, 4°C) to separate the cytosolic (supernatant) and microsomal (pellet) fractions. The P2 synaptosomal pellet was resuspended in 100 µl 4 mm HEPES buffer (mm) (4 HEPES, 1 EDTA, pH 7.4) and again centrifuged (12 000 × g, 20 min, 4°C). Resuspension and centrifugation was repeated. The resulting pellet was resuspended with buffer A (mM) (20 HEPES, 100 NaCl, 0.5% Triton, pH 7.2) and rotated slowly (15 min, 4°C), followed by centrifugation (12 000 × g, 20 min, 4°C). The supernatant (triton-soluble NP fraction) containing non-PSD membranes was retained. The pellet was resuspended in 120 µl buffer B (20 mm HEPES, 0.15 mm NaCl, 1% Triton-X, 1% deoxycholic acid, 1% SDS, 1 mm DTT, pH7.5), followed by gentle rotating (1 h, 4°C) and centrifugation (10 000 × g, 15 min, 4°C). The pellet was discarded and the supernatant (triton insoluble PSD fraction) retained. Microsomal, cytosolic, PSD and non-PSD samples were stored at −80°C until use. Protein concentration in the extracts was determined by Bradford assay (Bio-Rad). The extracts (500 μg protein) were incubated with nonspecific IgG (2 μg) or polyclonal rabbit anti-DAPK1 (2 μg; Cell Signaling Technology, #3008) or anti-Tau (2 μg, Millipore, MAB361) overnight at 4°C, followed by the addition of 40 μl of Protein G-Sepharose (Millipore, 16–266) for 3 h at 4°C. For the Co-IP experiments in HEK293 cells, the cells were lysed in IP buffer (mm) (20 Tris–HCl (pH 8.0), 100 NaCl, 1 EDTA, and 0.5% NP-40) containing protease inhibitor cocktail (Roche, 04693116001). After 20 min of incubation at 4°C with gentle vortex mixing, the cell lysates were centrifuged at 13 000 × g for 10 min at 4°C. Sample aliquots (500 μl) were precleared with 10 μl Protein A/G-agarose beads (11719394001, 11719386001, Roche) and incubated with 1 μg of antibody or control IgG overnight at 4°C, according to the manufacturer's recommendations. The precipitates were washed 4 times with lysis buffer and denatured with SDS sample buffer and separated by 12% SDS–PAGE. Proteins were transferred onto nitrocellulose membranes using a Bio-Rad mini-protein-III wet transfer unit overnight at 4°C. Transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in TBST buffer containing (mm): 10 Tris–HCl, 150 NaCl, and 0.1% Tween-20) for 1 h at room temperature, washed 3 times, and incubated with primary antibody against anti-DAPK1 (Cell Signaling Technology, #3008, 1:1000) or anti-Tau (Millipore, MAB361, 1:1000) for 1 h at room temperature. Membranes were washed 3 times with TBST buffer and incubated with the appropriate secondary antibodies for 1 h followed by washing 4 times. Signal detection was performed with an enhanced chemiluminescence kit (Amersham Biosciences). The lanes marked “input” were loaded with 10% of the starting material used for immunoprecipitation.
To determine the binding domain of DAPK1 with Tau protein, the DAPK1 deletion mutants (Flag-DAPK1ΔDD, Flag-DAPK1ΔKD, Flag-DAPK1K42A and Flag-DAPK1ΔCaM) were generated from full-length cDNA mouse DAPK11−1431. Purified Flag fusion proteins were separated using SDS–PAGE and transferred onto a nitrocellulose membrane, which was washed with distilled water and blocked with TBST for 1 h at room temperature. The membrane was then incubated with affinity binding buffer containing 50 mm Tris–HCl (pH 7.5), 200 mm NaCl, 12 mm-mercaptoethanol, 1.0% polyethylene glycol, 10 µg/ml protease inhibitors, and 500 µg/ml purified GFP-tagged Tau40 for 1 h at room temperature and washed 4 times for 5 min with affinity binding buffer. Bound DAPK1 and Tau40 was detected with anti-Flag (1:2000, Invitrogen) and anti-GFP (1:1000, Invitrogen), respectively.
Recombinant DNA Construction
The truncated DAPK1 constructs, including DAPK1ΔKD (residues 288–1430), DAPK1ΔCaM, DAPK1ΔDD (residues 1–1398), and DAPK1K42A mutants, were received as a gift from Prof. R-H Chen (Institute of Biological Chemistry, Academia Sinica). The vectors were re-cloned into the rAVE construct through ApaI/KpnI (GenDetect).
cDNA encoding Tau40 in pRK5 was used as the template for PCR amplification. The PCR products were digested using SalI and Cla1. The resulting fragment was ligated to an SalI-digested rAVE-eGFP vector (GenDetect, Ltd.) to generate rAVE-Tau40-eGFP under the control of a CAG enhancer and was terminated using the polyadenylation signal in the 3′ long terminal repeat. Phospho-deficient (substitution of serine at position 262 to alanine, S262A) construct was created by site-directed mutagenesis using the QuikChange Kit (Agilent Technologies) according to the supplier's manual and verified by sequencing analysis. Tau-S262A and Tau-WT (wild type) were subcloned into the rAVE-eGFP vector (GenDetect, Ltd.). rAVE-Tau40-eGFP or rAVE-Tau-S262A-eGFP was co-transfected with the AAV helper 1/2 into HEK293 cells to generate high titers (9 × 1012 genomic particles/ml) of rAAV1/2 infectious particles.
Phosphorylation Site Analysis by Mass Spectrometry
Phosphorylation can occur at a serine (S), threonine (T) or tyrosine (Y) residue. We identified phosphorylated residues in the Tau sequences using phosphorylated residue prediction tool, GPS2.1 (Xue et al. 2008). The prediction threshold value for GPS2.1 was set to be “high.” To further confirm the predicted phosphorylation sites, purified DAPK1-precipitated Tau protein samples were separated by SDS–PAGE gel. Corresponding areas of phosphorylated Tau (pTau) (40–70 kDa) were cut out from the coomassie blue stained SDS–PAGEs. Gel pieces underwent in-gel digestion and were subjected to a nanoLC-ESI-Iontrap mass spectrometer (BrukerDaltonik GmbH). Each spectrum was internally calibrated with mass signals of trypsin autolysisions to reach a typical mass measurement accuracy of ±10 ppm. Raw data were searched in the SWISS-PROT database.
Primary Neuron Culture and Plasmid Transfection
Cerebral cortex was isolated from the E20 wild-type mice or DAPK1-KD−/− mice, as we described before (Tu et al. 2010; Yang et al. 2012). Cells were dissociated and purified using papain dissociation kit (Worthington Biochemical Corporation) platted with the densities of 100–150 cells/mm2 on 19-mm coverslips coated with 30 μg/ml poly-d-lysine and 2 μg/ml laminin. Cells were placed in fresh serum-free Neurobasal Medium (21 103, GIBCO) plus 2% B27 and fed every 4 days with fresh media. The cultures were immunostained for β-tubulin III (Tuj1), a neuronal marker to confirm that these cultures were >85% neurons. On Day 9 (DIV 9), DAPK1 or DAPK1-KD−/− was co-expressed with a wild-type Tau (Tau-WT) or Tau-S262A mutant (S262A) in HEK293 cells using the respective rAAV1/2-virus particles, as indicated.
Whole-cell patch-clamp recordings were performed in voltage clamp mode using Axopatch 200B amplifier (Axon Instruments, Inc.), digitized at 10 kHz and filtered at 2 kHz. Data were acquired and analyzed using Clampfit 10.0 software (Axon Instruments, Inc.). Whole-cell recording pipettes (3–5 MΩ) were filled with a solution containing (in mm): 120 CH4SO3, 20 CaCl2, 4 NaCl, 10 HEPES, 0.05 EGTA, 4 Mg2ATP, 0.2 Na3GTP, 5 QX314 and ∼290 mOsm. The bath solution contained (in mm): 124.0 NaCl, 3.0 KCl, 26.0 NaHCO3, 1.2 MgCl2•6H2O, 1.25 NaH2PO4•2H2O, 10.0 C6H12O6, 2.0 CaCl2 (pH 7.4), ∼305 mOsm. Miniature AMPAR-dependent EPSCs were isolated by including D-APV (50 µm), bicuculine (10 µm), and 1 µm tetrodotoxin in the bath solution. All recordings were performed at a holding potential of −70 mV at room temperature. mEPSCs were identified using a template with a threshold of −6 pA (2.5 × SD of the noise) and were individually proofread for accuracy. To plot summary graphs, the average frequency from the eGFP control cells from each culture preparation was normalized. Individual cells from all 4 conditions were then compared with this normalized average.
Magnetic Resonance Imaging
Animals were anesthetized with isoflurane 1.5–2% and kept at 37°C, and an MR compatible respiration sensor was used to control the animals. All the magnetic resonance imaging (MRI) experiments were performed on a BIOSPEC BMT 47/40 (Bruker) spectrometer operating a 4.7T, equipped with an 11.2-cm actively shielded gradient system, capable of 200 mT/m gradient strength and 80 μs of rise time. A 7-cm bird cage radiofrequency coil was used for transmission and reception. T2-Weighted Imaging (T2WI) were acquired using a rapid acquisition with relaxation enhancement (RARE) technique, with a repetition time (TR) = 1650 s, RARE factor = 16, and inter echo interval = 10 ms, resulting in an effective echo time (TE) = 80 ms, the field of view (FOV) = 4 × 4 × 4 cm3. The acquired matrix size was 128 × 128 × 64. These data were zero-filled to obtain a reconstructed matrix size of 128 × 128 × 128.
Fluoro-Jade C Staining
The brain tissue slides were immersed for 3 min in 100% ethanol, for 1 min in 70% ethanol, and for 1 min in distilled water and then incubated in a solution containing 0.01% Fluoro-Jade C (Millipore) and 0.1% acetic acid (1:10) for 30 min on a shaker. After three 10-min washes, the slides were cover-slipped and imaged with a laser-scanning confocal microscope (LSM 510, Carl Zeiss). The number of FJ positive (FJ+) cells in each group was counted.
Golgi Staining and Synaptic Spine Analysis
Brains were removed from mice and immediately immersed in Solution A and B for 2 weeks at room temperature and transferred into Solution C for 24 h at 4°C, as instructed on the manufacture's experimental methods (FD rapid Golgi Stain kit, FD NeuroTechnologies). The brains were sliced using a Vibratome (VT1000S; Leica) at a thickness of 100 µm. Bright-field microscopy (Axio Observer; Zeiss) images were taken of cortex pyramidal neurons (50 cells with a total of 60 cm length of dendrites per group were analyzed). Images were coded and synaptic spines counted in software with Image Pro plus 6.0. All the spines were counted, and spine densities were expressed as spine/µm dendrite.
Administration of Peptides
The mice underwent a 60 min MCAO were injected (i.v.) with vehicle (saline) or TAT-R1D or TAT-s-R1D at a single dose of 0.5, 1, 2, 5, or 10 mg/kg body weight. In another group, 3, 6, or 9 h after MCAO, mice were injected with vehicle or TAT-R1D or TAT-s-R1D (2 mg/kg body weight, i.v.). The peptides were numbered and the experimenters were unaware of which one was applied in all experiments.
Neurological performance was scored daily using modified 7-point neurological scales (Tu et al. 2010); 0, no observable neurological deficits; 1, flexion of the contralateral torso; 2, circling to the ipsilateral side but normal posture at rest; 3, circling to the ipsilateral side; 4, rolling to the ipsilateral side; 5, leaning to the ipsilateral side at rest; 6, longitudinal spinning; and 7, no spontaneous motor activity/death. Score were ranked from 0 to 7 at an interval of 1.0. Neurological performance was always assessed by the blinded independent investigators.
Rotarod Treadmill Test
Motor coordination of the animals after operation and treatment was measured using a rotarod treadmill for mice (Ugo Basile), under the accelerating rotor mode (10 speeds from 4 to 40 rpm for 5 min). The interval from when the animal mounted the rod to when it fell off was recorded as the retention time, and the population of mice that walked for 300 s on the accelerating rotating rod was recorded as the walking survivor (Chen et al. 2012). The animals were trained for 2 days, 3 trials per day, before surgery, and the mean duration on the rod was recorded to obtain stable baseline values. Performance on the rotarod test was measured 3 times a day in the following 4 weeks after ischemic insult and peptide administration.
All variance values in the text and figure legends are represented as mean ± SEM. Western blot results, spine analysis data, infarct volumes, TUNEL or FJ staining positive cells, Neurological scores, and electrophysiology data were analyzed using the ANOVA with the Student–Newman–Keuls (SNK) tests. The data of behavioral studies were analyzed by two-way repeated-measures ANOVA with Bonferroni comparisons. Statistical analyses were performed using SPSS 13.0 (IBM) and Sigmaplot 12.0. Statistically significant differences were defined as P < 0.05.
Spine Damage Precedes to Apoptosis in Cerebral Ischemia
Dendritic spine density in the cortical neurons was analyzed in the frontal cortex of 2, 6, 12, and 24 h reperfusion after 1 h of MCAO by confocal imaging in brain tissue with AAV-eGFP infection 15 days before MCAO surgery (Fig. 1A). In total, 1560 spines were counted on 30 dendrites per group. Hereby, we detected significant decreases in dendritic spine density from 2 h to 24 h after MCAO (Fig. 1B,C). Consistently, the synaptic related proteins, postsynaptic density protein 95 (PSD95), AMPA receptor subunit GluR1 (GluA1), and Synapsin 1, were dramatically decreased 2 h after MCAO in the ischemic area (Fig. 1D,E). Simultaneously, the TUNEL-labeled (TUNEL+) apoptotic cells in the same brain area were examined at 2, 6, 12, and 24 h of reperfusion after 1 h MCAO. We observed notable increases of TUNEL+ cells from 6 h to 24 h after MCAO (Fig. 1F,G). In parallel, the protein levels of cleaved caspase 3 (Casp.3) were significantly increased 12 h after ischemic injury (Fig. 1H,I). Taken the above results together, we found the spine damage was prior to apoptosis in cerebral ischemia.
DAPK1 Interacts with Tau via its KD
To test whether DAPK1-Tau interaction mediates spine damage, we first examined whether DAPK1 interacts with Tau in ischemic brain tissue and primary cultured neurons under oxygen glucose deprivation (OGD). By double immunofluorescent staining and co-immunoprecipitation, we found that endogenous DAPK1 and Tau formed a complex in ischemic stroke (Fig. 2A–D). Though the expression of DAPK1 and Tau was not changed after ischemia (Supplementary Fig. S1A,B), the activity of DAPK1, which is reflected by phosphorylation of Myosin Light Chain (pMLC, a substrate of DAPK1 to reflect DAPK1 catalytic activity), was substantially increased 2 and 24 h after stroke onset (Supplementary Fig. S1C).
To learn how DAPK1 associates with Tau, we co-transfected GFP-tagged Tau (GFP-Tau) with Flag-tagged DAPK1 (Flag-DAPK1) into HEK293T cell. GFP-Tau might combine with Flag-DAPK1 (Fig. 2E) and form stable complexes. To identify the domain(s) in DAPK1 necessary for the interaction, we generated different DAPK1 mutants (Fig. 2F and SupplementaryFig. S2A,B). GST pull-down experiments revealed that GST-Tau, but not control GST, bound to Flag-DAPK1, its constitutively active form Flag-DAPK1ΔCaM, inactive form Flag-DAPK1K42A and DD deletion form Flag-DAPK1ΔDD, except DAPK1 KD deletion form Flag-DAPK1ΔKD (Fig. 2G). In addition, as shown by co-immunoprecipitation, all DAPK1 mutants could bind to Tau, except the DAPK1 KD deletion mutant (Flag-DAPK1ΔKD) (Fig. 2H,I and SupplementaryFig. S2C–E), suggesting that the KD is necessary for DAPK1 binding to Tau.
DAPK1 Phosphorylates Tau on Ser262 in Stroke
Given DAPK1 associated with Tau, a central question is whether DAPK1 phosphorylates Tau in vitro and in vivo. To determine whether Tau is a substrate of DAPK1 we directly assessed Tau phosphorylation site by the prediction software GPS 2.1 and nano-LC-ESI-MS/MS analysis. GPS 2.1 analysis suggested Ser262 of Human tau isoform (NCBI Reference Sequence: NP_005901.2) as a potential phosphorylation site for DAPK1 (Fig. 3A). Mass spectrum also demonstrated the Ser262 of Human tau isoform within the peptide IGSTENLK by DAPK1 after ischemic injury (Fig. 3B,C; Supplementary Fig. S2A, Supplementary Table S1 and S2). Blast results from the NCBI protein database revealed that the peptide IGSTENLK is highly conserved among different mammalian species (Supplementary Fig. S2B). No other Tau phosphorylation site than Ser-262 was identified in the Mass Spectrum assay (Supplementary Table S3).
To further validate Human Tau Ser262 as the phosphorylation site for DAPK1 in vitro, we generated a mutant form of Tau in which the phosphorylation site Serine 262 was mutated to Alanine 262 (S262A) and co-expressed DAPK1 in the HEK293 cells with S262A. Immunocytochemistry results demonstrated DAPK1 did not co-localize with S262A (Fig. 3D). In addition, co-immunoprecipitation experiments showed that DAPK1 did not interact with S262A (Fig. 3E). Western blottings also showed that the expression of pS262 was dramatically increased in parallel with cleaved caspase 3 in HEK293 cells transfected with DAPK1 and Tau-WT, but not DAPK1 and S262A (Fig. 3F).
To further investigate phosphorylation of Tau on Ser-262 after ischemia in vivo, we next subjected C57BL/6 mice with MCAO for 60 min and examined Tau phosphorylation. Western blotting results showed that pS262, but not previously reported pS202 or pS422, was detected. It is known that glycogen synthase kinase 3β (GSK3β) is a key regulatory kinase of Tau. Unexpectedly, neither GSK3β nor its activated form, phosphorylated GSK3β (pGSK3β), is significantly altered within 24 h after MCAO operation (Fig. 3G). These results indicate that DAPK1 phosphorylates Tau on Ser262 in cerebral ischemic stroke.
Phosphorylation of Tau on Ser262 Induces Spine Damage
Next, we co-transfected Tau-WT with DAPK1, S262A with DAPK1 into cultured primary cortical neurons or transfected Tau-WT into DAPK1-KD−/− cortical neurons from DAPK1-KD−/− mice to test whether Tau phosphorylation at Ser262 by DAPK1-KD mediates spine damage and neuronal apoptosis. Tau or S262A was fused with eGFP for visualization. DAPK1 and Tau-WT were co-transfected in cultured neurons at 9 days in vitro (DIV 9) (Fig. 4A). We found that 12 days after transfection, there was a significant decrease in spine density (Fig. 4B), an increase in synaptic pS262 (Fig. 4C), and a reduction in synaptic proteins including PSD95, GluA1, and Synapsin 1 (Fig. 4D). Consistently, we found a significant decline in both amplitude and frequency of AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) (Fig. 4E). On the contrary, co-transfection of DAPK1 with S262A or transfection of Tau-WT in DAPK1-KD−/− neurons decreased pS262 level and alleviated spine loss (Fig. 4B–E).
To further define the effect of pS262 on neuronal apoptosis after ischemic insults in vitro, we treated the primary cultured neurons with OGD. We found that the TUNEL+/pS262+ cells were significantly decreased 60 min after OGD treatment in S262A-transfected neurons compared with Tau-WT transfected neurons (Fig. 4F). Taken together, these results suggest that Tau is probably a major mediator of DAPK1 toxicity on dendritic spines and synaptic functions, which is largely dependent on phosphorylation of Ser262.
Genetic Deletion of DAPK1-KD Protects against Stroke Damage
To confirm the role of DAPK1-KD on spine damage and stroke injuries in vivo, we generated a conditional mutant strain of mice with a selective deletion of DAPK1-KD in the brain (DAPK1-KD−/− mice) by crossing CaMK2α-creERT2 and DAPK1-KD-Floxed transgenic mice (DAPK1-KDloxp/loxp mice) (Supplementary Fig. S4A). Deletion of KD induced 80% reduction in DAPK1-KD mRNA (Supplementary Fig. S4B) and >90% decrease of DAPK1 N terminal protein (Supplementary Fig. S4C). Furthermore, conditional knockout KD did not influence the animals' phenotype, such as body weight gain (Supplementary Fig. S4D,E), cerebral blood vessels and blood flow (Supplementary Fig. S4F), brain structure (Supplementary Fig. S4G), and anxiety-like behavior (Supplementary Fig. S4H). These results confirmed the successful deletion of KD in the brain, and this deletion exerted no significant phenotypic changes on mice.
Next, we subjected the DAPK1-KD−/−, and DAPK1-KDloxp/loxp mice with MCAO for 60 min and performed MRI studies 24 h after reperfusion. Magnetic resonance imaging revealed a marked reduction of infarction area (13.6 ± 4.2 mm3) when compared with the control DAPK1-KDloxp/loxp mice (47.8 ± 5.1 mm3) (Fig. 5A). Furthermore, immunoblotting of infarct brain tissue from the DAPK1-KD−/− mice showed lesser pS262, more PSD95, GluA1, and Synapsin1 24 h after ischemia than the DAPK1-KDloxp/loxp mice (Fig. 5B). Three days after reperfusion, frozen brain sections were prepared and stained with Fluoro-Jade C (FJ) to detect neuronal death, and an unbiased stereological analysis was adopted to measure the FJ-labeled (FJ+) area in the ischemic brain. Much less neuronal death was detected in DAPK1-KD−/− mice compared with DAPK1-KDloxp/loxp mice (Fig. 5C). The reduction of brain infarction area was associated with alleviated spine loss (Fig. 5D), improved neurological scores and motor coordination in DAPK1-KD−/− mice (Fig. 5E,F). These results further confirmed the above-mentioned conclusion that phosphorylation of Tau on Ser262 by DAPK1-KD induces spine damage after ischemia.
TAT-R1D Produces the Therapeutic Effects against Stroke Damage
Given that we have confirmed the interaction of DAPK1-KD with the microtubule repeat binding domain 1 of Tau (R1D) consisting of amino acids IGSTENLK, we hope to further explore the significance of the association. To this end, we generated a membrane-permeable peptide by fusing the peptide IGSTENLK to the transduction domain of the HIV TAT protein, named TAT-R1D. The HPLC graph and parameters of the purified peptide IGSTENLK are shown in Supplementary Figure S5A. Fluorescein isothiocyanate was fused to the terminal of TAT-R1D to visualize neuronal uptake of the peptide. We operated adult male mice with MCAO for 60 min followed by reperfusion. Three hours later, animals were intravenously (i.v.) administered with a single dose of 0.5, 1, 2, 5, or 10 mg/kg TAT-R1D, scrambled control peptide (TAT-s-R1D) or vehicle control (saline) (Fig. 6A, upper panel). Our data showed that intravenous injection-induced uptake of TAT-R1D into neuronal cells, but not microglia or astrocytes (Supplementary Fig. S5B). At a dose of 2 mg/kg (i.v.), TAT-R1D effectively disrupted the DAPK1-Tau association in brain tissues (Fig. 6B). To evaluate the intervention time window, we administered mice with TAT-R1D (2 mg/kg, i.v.) at 3 indicated time points (3, 6, and 9 h after reperfusion) (Fig. 6A, lower panel). Three and six hours after reperfusion, TAT-R1D was effective in dissociating DAPK1-Tau interaction (Fig. 6C). These results indicate that the 2 mg/kg (i.v.) may be used as an effective dose and 6 h after reperfusion may be taken for proper treatment time window.
To investigate the protective effects of TAT-R1D against spine damage after stroke, we examined the levels of pS262 and synaptic-related proteins. After treatment with TAT-R1D (2 mg/kg body weight, i.v.) 6 h after MCAO, immunoblotting of extraction of cortex tissue 24 h after MCAO showed inhibition in pS262 expression (Fig. 6D), prevention of the loss of both postsynaptic (PSD-95 and GluA1) and presynaptic proteins (Synapsin1) (Fig. 6E). Consistently, the reduction in both amplitude and frequency of AMPAR-mediated mEPSC after MCAO was rescued by TAT-R1D application (Fig. 6F). These results demonstrate that TAT-R1D is effective and efficient in blocking ischemia-induced spine damage.
To further confirm whether TAT-R1D might be used as a potential neuroprotective agent in stroke, MCAO surgery or photothrombosis (PT) by cold light illumination was performed to induce focal cerebral ischemia. Six hours after ischemia, mice were administered with TAT-R1D, TAT-s-R1D, or vehicle. The brain infarct was measured 3 days later with TTC staining. We found that TAT-R1D at a single dose of 2 mg/kg bodyweight (i.v.) sufficiently decreased the cerebral infarction compared with mice treated with TAT-s-R1D or vehicle (Fig. 6G and Supplementary Fig. S5C). Furthermore, TAT-R1D treatment markedly improved the overall behavioral functions including neurological score (N.S.) (Supplementary Fig. S5D), Morris water maze (Supplementary Fig. S5E), and open-field test (Supplementary Fig. S5F) analyzed 7 days post-ischemia. Together, our data demonstrate that blockade of DAPK1-Tau interaction by TAT-R1D administration may be a promising target for stroke treatment.
Stroke is one of the leading causes of disability and mortality; however, effective measures to minimize brain damage and improve recovery are still lacking. Evidence from a number of studies suggests that spine damage is a preceding neuropathological process contributing to neuronal injury after stroke. This drives us to search for new targets for neuroprotection in the earlier stage of stroke. In the present study, we provide the first evidence that DAPK1-Tau interaction mediates spine damage and neuronal death in cerebral ischemic stroke. Our work also uncovers specific Tau-directed kinase DAPK1, which causes spine damage by phosphorylating Tau at Ser-262 (pS262) in the brain with ischemic insults. More importantly, treatment with TAT-R1D (IGSTENLK) results in effective dissociation of DAPK1-Tau complexes, causing a significantly decrease in the level of pS262, and apparent alleviation of infarction area as well as the neurological deficits induced by cerebral ischemic stroke. Thus, blocking DAPK1-Tau interaction could be a promising target for developing potential therapy for ischemic stroke.
Spine loss is prior to neuronal loss in many neurodegenerative diseases, such as Alzheimer's Diseases, Parkinson Disease. Failure of synaptic transmission can be observed within minutes following hypoxia (Corcoran and O'Connor 2013). Our results are consistent with the previous report that spine damage is a preceding neuropathological process contributing to neuronal injury after stroke, possibly due to the high sensitivity of synaptic activity to alterations in energy supply (Khatri and Man 2013). For example, results from magnetic resonance spectroscopy related the changes in neuroenergetics revealed that more energy were required for functional activation to neurotransmitter cycling at the level of the synapse (Waldvogel et al. 2000; Foo et al. 2012). And the structural and functional integrity of synaptic transmission profoundly depends on a superior oxygen and glucose supply (Acker and Acker 2004).
Tau alteration, dysfunction, and extensive neuron loss have long been associated with several neurodegenerative diseases (Brandt et al. 2005). Abnormal phosphorylation of tau is considered one of the earliest signs of neuronal degeneration and appears to precede tau aggregation (Braak et al. 1994). Site-specific phosphorylation of tau can be regulated by concerted and sequential action of many protein kinases (Stoothoff and Johnson 2005). Among these protein kinases, glycogen synthase kinase-3beta (GSK-3β) was the most powerful tau protein kinase (Jope and Johnson 2004), which could induce the phosphorylation of tau protein at many sites, such as Ser199/202 (Leroy et al. 2002). Transient cerebral ischemia could induce the site-specific hyperphosphorylation of tau protein (Wen et al. 2004). Indeed, we only observed the phosphorylation of Ser262, but not Ser199/202 or other previously reported phosphorylation sites (pS202, pS214, pT231, and pS422), indicating the specificity of activation by DAPK1 in cortex after stroke. Changes in tau phosphorylation sites during cerebral ischemia and reperfusion are likely due to changes in the activities of specific phosphatases and kinases (Mailliot et al. 2000). In response to ischemia/reperfusion processes, phosphorylation or dephosphophrylation of tau protein appears to be dynamically regulated at different time phases (Burkhart et al. 1998; Mailliot et al. 2000). Additional studies including high-throughput mass spectrum analysis of phophorylated tau in the different areas of brain or at different time points after stroke, as well as detection of phosphatases or kinases enzyme activity are needed to deepen our understanding of the molecular mechanisms.
We provide several pieces of evidence supporting a causative role of Tau phosphorylation in mediating dendritic spine injuries in ischemic stroke. The phosphorylation of tau at Ser262 may result in the reduction of its affinity to microtubule protein and promote microtubule depolymerization (Biernat et al. 1993), which will inhibit axonal transport, destabilize neuronal cytoskeleton, and may contribute to the apoptotic process induced by this ischemia/reperfusion insult. In addition, abnormally phosphorylated tau protein is less likely to be hydrolyzed by the proteolylic enzymes and slow in degradation (Gong et al. 2000), resulting in the accumulation in the neurons and the possible formation of indissolvable tau. Those are the possible causes of the increasing of tau in dentritic spines and damage of synapes during ischemia/reperfusion. Taken together, these results demonstrate that tau protein is involved in the pathophysiological mechanism of cerebral ischemia, and therefore interrupting tau phosphorylation may become the target for the treatment of cerebral ischemia.
During ischemic stroke, DAPK1 is activated in the postsynaptic area and phosphorylates NMDA receptor NR2B protein at Serine-1303 and finally contributes to neuronal death (Tu et al. 2010). Our results show the DAPK1-mediated Tau phosphorylation is dependent on its KD and show a different mechanism with DAPK1-p53 interaction, in which DAPK1 associates with p53 DNA-binding motif with its DD and is dependent on the activity of DAPK1. Furthermore, DAPK1-p53-induced cell death is caused by converging the mitochondrial-dependent necrosis pathway and transcriptional-dependent apoptosis pathway (Pei et al. 2014). Whereas DAPK1-Tau-mediated cell death is mainly through the toxicity of phosphorylated tau and its subsequent damage to spines and synapses. Due to the distinct cell death mechanisms of DAPK1-p53 and DAPK1-Tau interaction, we speculate that Tau or p53 is likely to be the different downstream signal of DAPK1 and mediate respective cell death signals. Whether this is a consequence of DAPK1-dependent potentiation of NR2B-containing NMDARs or direct activation of the neuronal death signaling cascade remains to be investigated.
We found that blocking the interaction between DAPK1 and tau by TAT-R1D significantly reduced stroke-induced spine damage and neuronal injury. Furthermore, the transduction of TAT-R1D in the brain tissues occurred rapidly at 3 h after peptide injection and can produce treatment effects up to 6 h after stroke. The therapeutic time window for most neuroprotective agents in stroke is often <4 h following the onset of stroke. Since the blood brain barrier (BBB) often remains intact during the 4-h therapeutic window, any neuroprotective agent that is developed for stroke therapy must be able to cross the intact BBB if administered systemically (Joliot and Prochiantz 2004). In addition, targeted delivery remains a challenge with regard to the application of this technology because TAT fusion proteins administered systemically might also be distributed to other organs, such as liver, kidney, spleen, lung, bowel, and heart, which might cause unexpected side effects (Kilic et al. 2006). Fortunately, it has been found that the distribution of the TAT-RID in many organs has no apparent side effects in the mice (data not shown). Therefore, the fusion peptide TAT-R1D may be a promising agent for future research in stroke with potential clinical application.
In conclusion, our results demonstrate that phosphorylation of Tau at Serine 262 by the KD of DAPK1 mediates spine damage and the subsequent neuronal death in ischemic stroke. Blocking the interaction between DAPK1 and Tau by small peptide of Tau (IGSTENLK) reduces the level of pS262 and prevents spine damage and the followed ischemic brain injury. Thus, preventing the DAPK1-Tau pathway by Tat-R1D could be a potential strategy for treating cerebral ischemic stroke.
This work was supported by National Natural Science Foundation of China (Grants: 81130079 and 91232302 to Y.M.L.; 81271270 to Y.S.; 81200863 to L.P.) and China Postdoctoral Science Foundation (2013M540583 to L.P.). Funding to pay the Open Access publication charges for this article was provided by National Natural Science Foundation of China (81130079 and 91232302 to Y.M.L.).
We greatly appreciate Prof. Hengye Man from Boston University for revising our manuscript; Dr Yihao Yao and Dr Shun Zhang from Radiation Center of Tongji Hospital for the MRI assistance; and Prof. Ruey-Hwa Chen from Institute of Biological Chemistry, Academia Sinica (Taiwan) for presenting the truncated DAPK1 constructs. Conflict of interest: None declared.