Humanin protects cortical neurons from ischemia and reperfusion injury by the increased activity of superoxide dismutase.
ABSTRACT The neuroprotective effects of superoxide dismutase (SOD) against hypoxia/reperfusion (I/R) injury and of humanin (HN) against toxicity by familial amyotrophic lateral sclerosis (ALS)-related mutant SOD led us to hypothesize that HN might have a role to increase the activity of SOD, which might be involved in the protective effects of HN on neuron against Alzheimer's disease-unrelated neurotoxicities. In the present study, we found that 4 h ischemia and 24 h reperfusion induced a significant increase in lactate dehydrogenase (LDH) release, malondialdehyde (MDA) formation and the number of karyopyknotic nuclei (4',6-diamidino-2-phenylindole dihydrochloride nuclear dyeing) and a decrease in the number of Calcein-AM-positive living cells and cell viability. Pretreatment of the cells with HN led to a significant decrease in LDH release, MDA formation and the number of karyopyknotic nuclei, and an increase in the number of Calcein-AM-positive living cells and cell viability in neurons treated with I/R. We also found a significant decrease in SOD activity in neurons treated with I/R only, while pre-treatment with HN before I/R induced a significant increase in the activity of SOD as compared with the I/R group. Our findings implied that HN protects cortical neurons from I/R injury by the increased SOD activity and that the protective effect of HN on neurons against I/R is concentration-dependent.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Humanin (HN) inhibits neuronal death induced by various Alzheimer's disease (AD)-related insults via an unknown receptor on cell membranes. Our earlier study indicated that the activation of STAT3 was essential for HN-induced neuroprotection, suggesting that the HN receptor may belong to the cytokine receptor family. In this study, a series of loss-of-function tests indicated that gp130, the common subunit of receptors belonging to the IL-6 receptor family, was essential for HN-induced neuroprotection. Overexpression of ciliary neurotrophic factor receptor alpha (CNTFR) and/or the IL-27 receptor subunit, WSX-1, but not that of any other tested gp130-related receptor subunit, up-regulated HN binding to neuronal cells, whereas siRNA-mediated knockdown of endogenous CNTFR and/or WSX-1 reduced it. These results suggest that both CNTFR and WSX-1 may be also involved in HN binding to cells. Consistent with these results, loss-of-functions of CNTFR or WSX-1 in neuronal cells nullified their responsiveness to HN-mediated protection. In vitro-reconstituted binding assays showed that HN, but not the other control peptide, induced the hetero-oligomerization of CNTFR, WSX-1, and gp130. Together, these results indicate that HN protects neurons by binding to a complex or complexes involving CNTFR/WSX-1/gp130.Molecular biology of the cell 05/2009; 20(12):2864-73. · 5.98 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The relationship between lipid peroxidation and uptake of transferrin- free iron, Fe(II), by reticulocytes in an experimental system for studying membrane transport of Fe(II) was investigated by using free radical scavengers: BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), superoxide dismutase, alpha-tocopherol, propyl gallate and DPPD (N,N-diphenyl-1,4-phenylenediamine), and producers: t-butyl hydroperoxide, cumene hydroperoxide, H2O2 and aluminium carbonate. Measurements were made of MDA (malondialdehyde) and the rate of Fe(II) uptake from a sucrose solution buffered at pH 6.5 by Pipes. Most scavengers and producers used could increase or decrease only slightly the rate of Fe(II) uptake and some of them had no effect on Fe(II) uptake and MDA could not be detected at iron concentration of lower than 10 microM and incubation time of 20 min. At iron concentration of higher than 100 microM and incubation time of 4 h, there was the production of MDA which increased with the increment of iron concentration of incubation medium and BHT could inhibit the production of MDA. In addition, no difference was found in the rates of Fe(II) uptake in three experimental groups whose incubation medium was buffered by Pipes, Mops and Mes respectively. The results suggested that iron could induce free radical reaction under experimental conditions, especially at high concentration of iron and longer incubation time; however, at low concentration of iron (<10 microM) and the usual incubation time (20 min) free radical reaction was very slight and the extent of the reaction was not enough to damage the integrity and function of the membrane of reticulocytes, and that Fe(II) uptake by reticulocytes was not the result of free radical reaction and lipid peroxidation. It was therefore concluded that iron could not initiate its own membrane transport in rabbit reticulocytes by free radical reaction and lipid peroxidation and that the experimental system we used for studying membrane transport of Fe(II) is valid.Biochimica et Biophysica Acta 02/1996; 1310(3):293-302. · 4.66 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.Journal of Neuroscience 02/2002; 22(1):209-17. · 6.91 Impact Factor