Yuanjun Zhu

Neuroprotective Effect of TAT-14-3-3ε Fusion Protein against Cerebral Ischemia/Reperfusion Injury in Rats

Stroke is the major cause of death and disability worldwide, and the thrombolytic therapy currently available was unsatisfactory. 14-3-3ε is a well characterized member of 14-3-3 family, and has been reported to protect neurons against apoptosis in cerebral ischemia. However, it cannot transverse blood brain barrier (BBB) due to its large size. A protein transduction domain (PTD) of HIV TAT protein, is capable of delivering a large variety of proteins into the brain. In this study, we generated a fusion protein TAT-14-3-3ε, and evaluated its potential neuroprotective effect in rat focal ischemia/reperfusion (I/R) model. Western blot analysis validated the efficient transduction of TAT-14-3-3ε fusion protein into brain via a route of intravenous injection. TAT-14-3-3ε pre-treatment 2 h before ischemia significantly reduced cerebral infarction volume and improved neurologic score, while post-treatment 2 h after ischemia was less effective. Importantly, pre- or post-ischemic treatment with TAT-14-3-3ε significantly increased the number of surviving neurons as determined by Nissl staining, and attenuated I/R-induced neuronal apoptosis as showed by the decrease in apoptotic cell numbers and the inhibition of caspase-3 activity. Moreover, the introduction of 14-3-3ε into brain by TAT-mediated delivering reduced the formation of autophagosome, attenuated LC3B-II upregulation and reversed p62 downregulation induced by ischemic injury. Such inhibition of autophagy was reversed by treatment with an autophagy inducer rapamycin (RAP), which also attenuated the neuroprotective effect of TAT-14-3-3ε. Conversely, autophagy inhibitor 3-methyladenine (3-MA) inhibited I/R-induced the increase in autophagic activity, and attenuated I/R-induced brain infarct. These results suggest that TAT-14-3-3ε can be efficiently transduced into brain and exert significantly protective effect against brain ischemic injury through inhibiting neuronal apoptosis and autophagic activation.

w007B protects brain against ischemia–reperfusion injury in rats through inhibiting inflammation, apoptosis and autophagy

This study was designed to investigate the effect of w007B, a newly synthesized derivative of honokiol, on MCAO reperfusion, and its therapeutic time window and related mechanisms in rats. Neurological deficit scores, infarct size and brain water content were measured after 24 h reperfusion following 2 h ischemia. The results showed that w007B (10 and 50 μg/kg, IV immediately after reperfusion) markedly decreased neurological deficit scores, reduced infarct size and alleviated brain water content, and then 50 μg/kg w007B given within 3 h after reperfusion (5 h after ischemia) significantly attenuated ischemia-induced brain injury. Additionally, no sign of toxicity was observed when a single dose of 50mg/kg w007B (1000 times of the highest effective dose, IP) was administered. To explore the underlying mechanisms, the expression level of apoptosis, inflammation and autophagy-related markers in brain tissue were detected with kits or by western blot. It was observed that w007B rapidly and significantly reduced caspase-3 activity and NO production in the injured semi-brain, and also lowered the level of the p65 subunit of NF-κB in the nucleus. Besides, it also reduced the expression of Beclin-1 and LC3B-II, and increased the level of p62, the autophagy-related proteins in I/R-injured hemisphere. In conclusion, w007B exerts neuroprotective effect on cerebral ischemia-reperfusion injury with wider therapeutic time window and better safety; its mechanisms may be associated with its anti-inflammation, anti-apoptosis and anti-autophagy action. These results suggest that w007B shows strong potential as a clinical neuroprotective candidate for the treatment of ischemic stroke.

Exogenous hydrogen sulfide attenuates cerebral ischemia-reperfusion injury by inhibiting autophagy in mice.

Hydrogen sulfide (H2S), the third gas transmitter, has been proven to be neuroprotective in cerebral ischemic injury, but whether its effect is mediated by regulating autophagy is not yet clear. The present study was undertaken to explore the underlying mechanisms of exogenous H2S on autophagy regulation in cerebral ischemia. The effects and its connection with autophagy of NaHS, a H2S donor, were observed through neurological deficits and cerebral infarct volume in middle cerebral artery occlusion (MCAO) mice; autophagy-related proteins and autophagy complex levels in the ischemic hemisphere were detected with Western blot assay. Compared with the model group, NaHS significantly decreased infarct volume and improved neurological deficits; rapamycin, an autophagy activator, abolished the effect of NaHS; NaHS decreased the expression of LC3-II and up-regulated p62 expression in the ischemic cortex 24 h after ischemia. However, NaHS did not significantly influence Beclin-1 expression. H2S has a neuroprotective effect on ischemic injury in MCAO mice; this effect is associated with its influence in down-regulating autophagosome accumulation.

Rational Design of Fluorescent Phthalazinone Derivatives for One- and Two-Photon Imaging.

Phthalazinone derivatives were designed as optical probes for one- and two-photon fluorescence microscopy imaging. The design strategy involves stepwise extension and modification of pyridazinone by 1) expansion of pyridazinone to phthalazinone, a larger conjugated system, as the electron acceptor, 2) coupling of electron-donating aromatic groups such as N,N-diethylaminophenyl, thienyl, naphthyl, and quinolyl to the phthalazinone, and 3) anchoring of an alkyl chain to the phthalazinone with various terminal substituents such as triphenylphosphonio, morpholino, triethylammonio, N-methylimidazolio, pyrrolidino, and piperidino. Theoretical calculations were utilized to verify the initial design. The desired fluorescent probes were synthesized by two different routes in considerable yields. Twenty-two phthalazinone derivatives were synthesized and their photophysical properties were measured. Selected compounds were applied in cell imaging, and valuable information was obtained. Furthermore, the designed compounds showed excellent performance in two-photon microscopic imaging of mouse brain slices.

Increased autophagic degradation contributes to the neuroprotection of hydrogen sulfide against cerebral ischemia/reperfusion injury

Hydrogen sulfide (H2S), an endogenous gaseous signal molecule, exhibits protective effect against ischemic injury. However, its underlying mechanism is not fully understood. We have recently reported that exogenous H2S decreases the accumulation of autophagic vacuoles in mouse brain with ischemia/reperfusion (I/R) injury. To further investigate whether this H2S-induced reduction of autophagic vacuoles is caused by the decreased autophagosome synthesis and/or the increased autophagic degradation inautophagic flux, we performed in vitro and in vivo studies using SH-SY5Y cells for the oxygen and glucose deprivation/reoxygenation (OGD/R) and mice for the cerebral I/R, respectively. NaHS (a donor of H2S) treatment significantly increased cell viability and reduced cerebral infarct volume. NaHS treatment reduced the OGD/R-induced elevation in LC3-II (an autophagic marker), which was completely reversed by co-treatment with an autophagic flux inhibitor bafilomycin A1 (BafA1). However, H2S did not affect the OGD/R-induced increase of the ULK1 self-association and decrease of the ATG13 phosphorylation, which are the critical steps for the initiation of autophagosome formation. Cerebral I/R injury caused an increase in LC3-II, a decrease in p62 and the accumulation of autophagosomes in the cortex and the hippocampus, which were inhibited by NaHS treatment. This H2S-induced decline of LC3-II in ischemic brain was reversed by BafA1. Moreover, BafA1 treatment abolished the protection of H2S on the cerebral infarction. Collectively, the neuroprotection of exogenous H2S against ischemia/hypoxia and reperfusion/reoxygenation injury is mediated by the enhancement of autophagic degradation.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Structure-guided creation of AcAP5-derived and platelet targeted factor Xa inhibitors

Anticoagulants and anti-platelet agents are simultaneously administrated in clinical practice (i.e. percutaneous coronary intervention), which cause significant risk of systemic bleeding. Targeted delivery of anticoagulants to the activated platelets at sites of vascular injuries may condense the site-specific anticoagulant effect and reduce the hemorrhage side effects in uninjured vessels. To this end, we prepared three ancylostoma caninum anticoagulant peptide 5 (AcAP5) variants NR1, NR2 and NR3 engineered with a platelet-binding Arg-Gly-Asp (RGD) motif and evaluated their anti-Factor Xa (FXa) and platelet-binding effects. These RGD-containing AcAP5 variants were capable of interacting with platelet receptor αIIbβ3 as shown in computational analysis. All variants, especially NR2 and NR3, retained entirely the anti-FXa function of parent AcAP5. Moreover, they prevented the formation of occlusive thrombi in rat carotid artery injury model, suggesting that they inhibit platelet aggregation in vivo. Further functional investigation of NR3 demonstrated that NR3 inhibited platelet aggregation in vitro and FXa activity in vivo, and prolonged the coagulation time, all in a dose-dependent manner. Through flow cytometry assay, we confirmed the binding of NR3 to αIIbβ3 receptor. In mouse model of carotid artery endothelium injury, NR3-treated mice showed less tail bleeding time than AcAP5-treated mice, and aspirin plus NR3 treatment exhibited moderate reduction of blood loss compared with aspirin plus AcAP5 treatment. These results indicate the feasibility to engineer a novel FXa inhibitor specifically targeting the activated platelets, which centralizes its anticoagulation efficacy in the injured vascular endothelium and reduces the risk of systemic bleeding.

Design and evaluation of EphrinA1 mutants with cerebral protective effect

The activation of EphA2 receptor by its natural ligand EphrinA1 causes blood brain barrier dysfunction, and inactivation of EphA2 reduces BBB damage in ischemic stroke. Thus, EphA2 targeted antagonists may serve as neuroprotective agents. We engineered four mutants of EphrinA1, EM1, EM2, EM3 and EM4, respectively. The computational analysis showed that these four mutants were capable of interacting with EphA2. Their potential neuroprotective effects were examined in mouse focal ischemia/reperfusion (I/R) model. EM2 exhibited strong neuroprotective effects, including reduced brain infarct volume, neuronal apoptosis, cerebral edema, and improved neurological scores. The EM2-mediated protection was associated with a comparative decrease in BBB leakage, inflammatory infiltration, and higher expression levels of tight junction proteins, such as zonula occludens-1 and Occludin. I/R-induced high expression of Rho-associated protein kinase 2 (ROCK2) was down-regulated after EM2 treatment. Moreover, EM2 reduced agonist doxazosin-induced EphA2 phosphorylation and cells rounding in PC3 cells, indicating EphA2-antagonizing activity of EM2. These finding provided evidences of the neuroprotection of EphA2 antagonist and a novel approach for ischemic stroke treatment. These results also suggested that a receptor agonist can be switched to an antagonist by substituting one or more relevant residues.

Engineering Novel Anticoagulant Proteins by Motif Grafting

Structure-based rational design has been considered as a promising approach to design novel proteins. For this purpose, we designed artificial anticoagulant proteins that are able to target Factor Xa (FXa) using a functional motifgrafting approach. The motif corresponded to the residues Cys15 to Cys42 of Ancylostoma caninum anticoagulant peptide 5 (AcAP5), a potent FXa inhibitor. By screening of the Protein Data Bank (PDB) using Vector Alignment Search Tool (VAST, search for three-dimensional scaffolds in protein structures), we screened scaffolds as hosts to reproduce the functional topology of this motif. Three designed artificial chimeric proteins were expressed and purified to test their FXainhibiting ability. One of the recombinant proteins, pep3, was found to inhibit FXa with strong activity (IC50 of 152 nM) in vitro. Moreover, pep3 inhibited arterial thrombosis formation in rats with uniform potency compared with natural AcAP5. Therefore, our data demonstrate that motif-grafting is a useful tool to engineer novel artificial anticoagulant proteins.

Revelation of the dynamic progression of hypoxia-reoxygenation injury by visualization of the lysosomal hydrogen peroxide

Hydrogen peroxide (H2O2) plays an important role in pathological conditions, such as cerebral ischemia-reperfusion (I-R) injury. Fluorescent probes may serve as valuable tools to detect the amount, temporal and spatial distribution of H2O2 in living cells. To investigate the role of lysosomal H2O2 involved in cerebral I-R injury, we designed and synthesized a lysosome-targetable two-photon fluorescent probe ztl-4, through expansion and substitution of the original pyridazinone scaffold, conjugation of electronic-donating aromatic ring and precise terminal modification of the alkyl linker. The probe ztl-4 exhibited fast, sensitive and highly selective response toward H2O2. ztl-4 could image exogenous H2O2 in SH-SY5Y cells and brain slices. In addition, ztl-4 was located in lysosomes with high colocalization coefficient compared with LysoTracker. ztl-4 was further applied for detecting the endogenous generation of H2O2 in SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) or OGD/reoxygenation (OGD/R) injury. Both OGD- and OGD/R-induced cell injury caused a time-dependent increase of H2O2 production within lysosomes. Moreover, OGD/R-treated cells showed much more amount of H2O2 than OGD-treated cells, indicating that reoxygenation will promote H2O2 accumulation in lysosomes of post-hypoxia cells. Therefore, the probe is suitable for monitoring the dynamic changes of lysosomal H2O2 in cells.