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.

Effects of low doses of Tat-PIM2 protein against hippocampal neuronal cell survival

“…PTDs have the ability to traverse the plasma membrane and enter cells on their own without the aid of a specific receptor [18,19]. Tat PTD has been extensively studied and demonstrated the ability to transduce into various cells and in various animal models [20][21][22][23]. We also demonstrated the protective effects of PTDs fusion protein against cell death in vitro and in vivo [24][25][26][27][28].…”

“…PTDs are small peptides with the ability to promote the functions of full-length proteins and enter all tissues including various cells and animal brain. Numerous studies have demonstrated the therapeutic potential of using PTD fusion proteins for the treatment of a variety of diseases [18][19][20][21][22][23][24]. After purified Tat-PIM2 protein was confirmed, we determined transduction ability of Tat-PIM2 protein into HT22 hippocampal neuronal cells.…”

Design and evaluation of EphrinA1 mutants with cerebral protective effect

“…The initial dosage of recombinant proteins was 10 mg/kg according to previous researches 16–18 . Vehicle-treated mice developed severe injuries with infarct volumes; while EM2 significantly reduced the infarct size and improved behavior score (Fig.…”

Growth factor therapy sequesters inflammation in affording neuroprotection in cerebrovascular diseases

“…Engineered proteins, such as the TAT-linked proteins, have been altered with polyethylene glycol (PEG) and functional peptides to increase the delivery efficiency [136]. The HIV TAT peptide, for example, has been linked to therapeutic proteins to allow efficient translocation across the BBB for ischemic stroke therapy [137]. Intravenously infused PEGylated-hemoglobin (PEG-hemoglobin), for example, has been evaluated in stroke and TBI animal models in which several advantages have been observed including increased stability and circulation time of drugs and increased diffusion to target sites [136,138].…”