title: Neuroprotective effect of TAT-14-3-3ε fusion protein against cerebral ischemia/reperfusion injury in rats.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China.
authors.forename: Yuanjun
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China.
authors.forename: Qixin
authors.initials: Q
authors.lastname: Bu
authors.affiliation: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Neuroscience, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China.
authors.forename: Yinye
authors.initials: Y
authors.lastname: Wang
mesh_terms: D048948:14-3-3 Proteins / Q000008:administration & dosage*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002540:Cerebral Cortex / Q000098:blood supply / Q000187:drug effects / Q000473:pathology; D004353:Drug Evaluation, Preclinical; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy*; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage*; D015427:Reperfusion Injury / Q000517:prevention & control*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage*
chemical_list: D048948:14-3-3 Proteins; D018696:Neuroprotective Agents; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus