| Abstract No.: | C-C3108 |
| Country: | Canada |
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| Title: | IDENTIFICATION OF HEME OXYGENASE-1 AS A NOVEL TARGET OF NEUROPROTECTION BY MINOCYCLINE IN HUMAN NEURONS |
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| Authors/Affiliations: | 1 Shuhong Liu*; 1 Mengzhou Xue; 1 V. Wee Yong;
1 University of Calgary, Edmonton, AB, Canada
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| Content: | Minocycline has been reported to have neuroprotective activity in various animal models of neurological diseases, where a major mechanism for its effectiveness is attributed to the inhibition of microglia inflammation. Much less has been described on the potential direct actions of minocycline on neurons independent of microglia activity.
Our aims are: 1) to test the hypothesis that minocycline could be neuroprotective without the intermediary of microglia; 2) to identify new targets of minocycline using microarrays; 3) to study the roles of heme oxygenase-1 (HO-1) in minocycline protection using both in vitro and in vivo experiments.
Materials and methods: HFNs were pre-treated with minocycline before the treatment with the oxidative agent, hydrogen peroxide (H2O2). Relative intensity of neuronal markers was then measured with In-Cell western analysis. Gene expression profiles of the treated HFNs were generated using microarrays. Real-time PCR (RT-PCR) was also conducted to validate the microarray results.
Results: Using cultured human fetal neurons (HFNs) which contain 85 to 90% neurons and 10-15% astrocytes, but devoid of microglia, we found that the oxidative agent, hydrogen peroxide (H2O2), kills HFNs which was documented by obvious cell loss, and by a decrease in the intensity of neuronal markers and active caspase-3 in In-Cell western analyses. The presence of minocycline significantly attenuated the effects of H2O2. As neurons could therefore be protected by minocycline in the absence of microglia, we subjected HFNs to DNA microarrays using a 14,000 gene chip. Across 14 experiments involving several different HFN preparations, we found that many genes were up-regulated by H2O2 and that a minority of these were reduced in expression levels when neurons were protected by minocycline. Of the latter targets, real time PCR and In-Cell western investigations confirmed that the oxidant modulating protein, HO-1, was up-regulated by H2O2 and reduced by minocycline. Immunohistochemistry for HO-1 showed that neurons and astrocytes were both reactive for this enzyme following H2O2 exposure. The necessity of HO-1 in minocycline protection against H2O2 is currently under investigation using both in vitro and in vivo experiments.
Conclusions: We conclude that minocycline confers protective effect of HFNs against H2O2 without the need of microglia activity and that this may involve a hitherto unknown mechanism of minocycline, that of modulating HO-1 levels in neurons and/or astrocytes.
*This research is supported by a grant from the Canadian Institutes of Health Research.
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