Novel Mechanisms Of Synaptic Damages In Ischemic Stroke Via DAPK1-mediated Death Signals

BackgroundIschemic stroke is due to disruption of cerebral blood flow, resulting in a serious neurological disease of cerebral ischemia. The patient will appear language impairment, visual loss, paralysis and even death. With the population of aging increasing, the high rate of morbidity, mortality and disability of stroke is rising gradually. At present, treatment for cerebral ischemia damage is very limited, the only effective one is the use of tissue plasminogen activator (tPA) thrombolytic therapy. However, because of security concerns and the narrow thrombolysis time window (<4.5hours), most patients receive only symptomatic and supportive cure. Therefore, to explore the intervention methods and new treatments against brain injury are extremely important.Synaptic damage is the initial pathophysiological event after stoke. The underlying mechanisms are still unclear. Death associated protein kinase (DAPK1), as a kind of Ca+/calmodulin dependent serine/threonine kinase, is activated in ischemic cerebral area in ischemic stroke, and mediates neuronal death. Cytoskeletal protein microtubule associated protein Tau, richly expressing in neuronal soma and neurite, maintains axonal transport, neuronal morphology, the signal communication between neurons through promoting microtubule assembly. Tau has lots of serine/threonine residues in the amino acid sequence. Many serine/threonine kinases (including DAPK1) are involved in tau abnormal hyperphosphorylation. But whether the activated DAPK1phosphorylates tau to induce neuronal death after ischemic stroke is unclear. Caytaxin richly expresses at the presynapses and involves in presynaptic damage in various neural diseases. Whether activated DAPK1interacting with Caytaxin and mediating presynaptic apoptosis in ischemic stroke remain unknown.Objective(1)Elucidate the cellular and molecular mechanisms of neuronal death after ischemic stroke. Provide evidence that Tau and Caytaxin are downstream substrates of DAPK1involved in the specific role of ischemic stroke. The activated DAPK1induced by ischemic stroke causes Tau and Caytaxin phosphorylated, resulting in loss of dendritic spines and synaptic dysfunction, and eventually establishes the basis for the structure and function of neuronal apoptosis.(2)Test whether Synthetic small molecular polypeptide TAT-R1Dcan block the interaction between DAPK1with Tau. Investigate the treatment strategy for ischemic stroke, which may lay a theoretical foundation for drug research and development for the treatment of ischemic stroke.Methods4-month-old C57BL/6J, CaMK II a-Cre, DAPK1-KDloxp/loxp, DAPK1-KD-/-were used in the following experiments. Middle cerebral artery occlusion (MCAO) and photothrombosis ischemia models were used to construct focal cerebral ischemia or sham control. Magnetic resonance images (MRI) and TTC staining were used to detect the ischemic area. Fluoro-Jade C and TUNEL staining were applied to label the number of degenerative and apoptosis neurons after ischemia respectively. Infection of AAV-EGFP was used to label spine of dendrites of neuron. Western blot was applied to detect the expression level of DAPK1, pMLC, Tau, Caytaxin, Cleaved-Caspase3, synaptic associated protein PSD95, GluRl and so on in cortical neurons after MCAO. Double immunofluorescence and co-immunoprecipitation on brain tissue after ischemia and primary neuron culture after OGD or H2O2treatment were used to identify the interaction of DAPK1and Tau, DAPK1and Caytaxin. Constructed expression system in HEK293T cells, and co-transfected DAPK1different mutants(DAPK1△KD, DAPK1△CaM, DAPK1△DD, DAPK1K42A) and Tau to identify the domain of DAPK1interacting with Tau. Mass spectrometric detection was used to conform phosphorylated proteins immunoprecipitated by DAPK1in cortical tissue after MCAO.Infected rAAV-Tau-WT, rAAV-Tau-S262A in DAPK1-KD+/+primary neurons and infected rAAV-Tau-WT in DAPK1-KD-/-primary neurons to observe the neuronal dendritic spines injury. Intravenous injection of TAT-R1D was used to observe whether it rescue stroke injury. Behavior detection methods such as water morris maze and open field test were applied to view learning and memory and the activities of mice after ischemia or not.ResultsSpine damage precedes to apoptosis in cerebral ischemia. Significant decreases were detected in dendritic spine density from2hours to24hours after MCAO.The synaptic related proteins, PSD95, GluAl and Synapsin1, dramatically decreased2hours after MCAO in the ischemic area. Notable increases were observed in TUNEL+cells from6hours to24hours after MCAO. In parallel, the protein levels of cleaved caspase3(Casp.3) were significantly increased12hours after ischemic injury.DAPK1interacts with Tau via its kinase domain, and DAPK1interacts with Caytaxin in presynaptic area. Endogenous and exogenous DAPK1and Tau formed a complex in ischemic stroke and cell line expression system as same as DAPK1and Caytaxin, though the expression of DAPK1and Tau did not change while Caytaxin increased after ischemia. GST pull-down experiments and constructing expression system in HEK293T revealed that GST-Tau, bound to Flag-DAPK1, Flag-DAPK1△CaM, Flag-DAPK1K42A and Flag-DAPK1ADD, except Flag-DAPK1AKD. DAPK1and Caytaxin both expressed richly in presynaptic area, and showed an enhanced interaction in brain after ischemic stroke and in primary neurons after H2O2treatment.DAPK1phosphorylates Tau on Ser262, phosphorylates Caytaxin on Ser46in ischemic stroke. GPS2.1analysis suggested Ser262of Human Tau isoform as a potential phosphorylation site for DAPK1. Mass spectrum confirmed it. And it also demonstrated the Caytaxin Ser46was phosphorylated by DAPK1after ischemic injury. Co-immunoprecipitation showed that DAPK1did not interact with S262A. Western blottings also showed that the expression of pS262was increased in parallel with Cleaved Caspase3in HEK293cells transfected with DAPK1and Tau-WT, but not DAPK1and S262A. Tau pS262was increased instead of the previously reported pS202or pS422. And glycogen synthase kinase3β(GSK3β), a key regulatory kinase of Tau, was not altered after MCAO.Phosphorylation of Tau on Ser262induces spine damage. There was a significant decrease in spine density, an increase in synaptic pS262, a reduction in synaptic proteins including PSD95, GluAl, and Synapsin1, and a significant decline in both amplitude and frequency of AMPAR-mediated miniature excitatory postsynaptic current (mEPSC)12days after infection Tau-WT viruses in cultured DAPK1-KD+neurons at9days in vitro. On the contrary, there was not in infection of Tau-S262A in DAPK1-KD+/+neurons or infection of Tau-WT in DAPK1-KD-/-neurons. The TUNEL+/pS262+cells were decreased60min after OGD treatment in Tau-S262A infected neurons compared with Tau-WT.Genetic Deletion of DAPK1-KD Protects Against Stroke Damage. DAPK1-KD/-mice did not show any abnormal phenotypes compared with its wide type lirtermates. MRI and TTC staining revealed a marked reduction of infarction area24h after MCAO, FJ and TUNEL staining showed much less neuronal death3d and7d after MCAO, immunoblotting of infarct brain tissue showed less pS262, more PSD95, GluAl and Synapsin Ⅰ24hours after MCAO, as compared with the control mice. Alleviated spine loss, improved neurological scores and motor coordination were seen in DAPK1-KD-/-mice.TAT-R1D Produces Therapeutic Effects Against Stroke Damage. Intravenous injection induced uptake of TAT-R1D, a peptide synthetized based the interaction of DAPK1and Tau, into neuronal cells, but not microglia or astrocytes. TAT-R1D effectively disrupted the DAPK1-Tau association in brain tissues. Treatment with TAT-R1D (2mg/kg body weight,i.v.)1hours after MCAO, showed an inhibition in pS262expression and prevention the loss of synaptic associated proteins, reduced the cerebral infarction, improved the overall behavioral functions including neurological score, morris water maze and open field test compared with treatment of TAT-s-R1D or vehicle.ConclusionsWe provide the first evidence that DAPK1-Tau interaction mediates spine damage and neuronal death in cerebral ischemic stroke. Our work also uncover specific Tau-directed kinase DAPK1, which causes spine damage by phosphorylating Tau at Ser-262(pS262) in the brain with ischemic insults. More importantly, either genetic deletion of DAPK1kinase 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. Moreover, the interaction between DAPK1and Caytaxin is extremely likely to mediate presynaptic dysfunction after ischemic stroke. Thus, disruption of DAPK1-Tau and DAPK1-Caytaxin interaction could be a promising target for ischemic stroke trement.