Selectively increasing inducible heat shock protein 70 via TAT-protein transduction protects neurons from nitrosative stress and excitotoxicity.
Induction of heat shock protein 70 (Hsp70) via sublethal stress protects neurons from subsequent lethal injuries. Here we show that specific and efficient intracellular transduction of Hsp70 can be achieved utilizing an 11 amino acid leading sequence from human immunodeficiency virus (TAT-Hsp70) in primary neuronal cultures. Western blot and immunohistochemistry demonstrated intracellular accumulation of Hsp70 in insoluble protein fractions and mitochondrial compartments. We then examined the effects of Hsp70 overexpression using TAT-Hsp70 in models of nitrosative and excitotoxic neuronal death in vitro. Neurons were pre-incubated with 300 nM TAT-Hsp 70 overnight, then exposed to either peroxynitrite (ONOO-) or glutamate. TAT-Hsp70 maintained cellular respiration, inhibited extracellular lactate dehydrogenase release, and/or reduced cell death assessed by flow cytometry vs. vehicle, wild-type Hsp70, and TAT-beta-galactosidase controls. Hsp70 transduction using a TAT fusion protein is an effective method to selectively increase Hsp70 in neurons and is sufficient to provide neuroprotection from nitrosative stress and excitotoxicity. Further study is needed to confirm whether TAT-Hsp70 is protective in in vivo models of brain injury.
Department of Critical Care Medicine, The Safar Center for Resuscitation Research, University of Pittsburgh, Pennsylvania, USA.
Yichen
Y
Lai
Lina
L
Du
Katherine E
KE
Dunsmore
Larry W
LW
Jenkins
Hector R
HR
Wong
Robert S B
RS
Clark
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods; D015696:Gene Products, tat / Q000502:physiology*; D018698:Glutamic Acid / Q000633:toxicity*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D045022:Lactate Dehydrogenases / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D030421:Peroxynitrous Acid / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D014161:Transduction, Genetic / Q000379:methods*
D015696:Gene Products, tat; D018840:HSP70 Heat-Shock Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D030421:Peroxynitrous Acid; D018698:Glutamic Acid; D045022:Lactate Dehydrogenases; C022616:thiazolyl blue