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.
Highlights
Stroke is responsible for more than 5 million deaths each year worldwide, making it the second leading cause of death and a major cause of disability [1]
We further examined the neuroprotective effect of TAT-14-3-3e against rat transient focal cerebral ischemia
14-3-3e in ischemic brain damage, we examined the effect of TAT-14-3-3e on ischemia/ reperfusion (I/R)-induced autophagy by Transmission Electron Microscopy (TEM) analysis and Western blot analysis with autophagy specific antibodies against microtubule-associated protein 1A light chain 3 (LC3), autophagerelated gene 6 (Atg-6)/Beclin-1 and p62/SQSTM1
Summary
Stroke is responsible for more than 5 million deaths each year worldwide, making it the second leading cause of death and a major cause of disability [1]. This situation is becoming worse in the aged due to the increasing prevalence of some risk factors, like hypertension, coronary artery disease and atherosclerosis [2], [3]. Thrombolytic therapy is the only approved treatment for acute ischemic stroke [4], [5]. Neuroprotective approaches, as prospective newer treatments for stroke, have shown promise in animal models in recent decades, but their efficacies in patients remain limited [7]. There is an urgent need to develop effective neuroprotective agents to prevent and treat stroke
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