PEP-1-metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults.

BACKGROUND: Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult.
METHODS: A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1-MT-III protein. PEP-1-MT-III protein was purified using affinity chromatograph. Transduced PEP-1-MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry.
RESULTS: Purified PEP-1-MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1-MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1-MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1-MT-III protein protects against oxidative stress induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE: Transduced PEP-1-MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1-MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.