Most vertebrate brains are exquisitely sensitive to low oxygen levels and respond to this hypoxia with a variety of protective mechanisms to increase anaerobic metabolism and oxygen delivery, minimizing the pathological effects of deficient energy supplies. From the initial reports of their existence several years ago, the heme proteins neuroglobin and cytoglobin have been the subjects of much study: able to bind oxygen reversibly, these proteins could act as neuronal and tissue `myoglobins', yet their true roles are unclear. Possible functions include serving as an intracellular oxygen carrier or oxygen sensor, or involvement in the metabolism of nitric oxide or reactive oxygen species (ROS) generated when hypoxic cells are reoxygenated. Extensive ROS generation damages cells by oxidizing lipids, proteins and DNA. Thus,compounds that decrease ROS aid in cell survival, and it has been shown that the over-expression of neuroglobin or cytoglobin promotes cell survival after hypoxia or ischemia.
Sylvia Dewilde and her colleagues at the University of Antwerp investigated the function of these heme proteins by either over-expressing or under-expressing (using antisense treatment) cytoglobin and neuroglobin in human neural cells in cell culture and exposing the cells to either oxygen deprivation (anoxia), or oxygen and glucose deprivation. They then compared cell survival and production of the ROS hydrogen peroxide(H2O2), as well as mRNA and protein levels in the cell groups.
The unmanipulated cells easily tolerated as much as 32 h anoxia, though when deprived of glucose and oxygen, cell death began to increase after 12 h and only 46% of cells survived 32 h, showing that neuronal cells are far more vulnerable to cell death when both oxygen and glucose are absent. To investigate how an excess of heme proteins affected cell survival, the team over-expressed neuroglobin or cytoglobin in some cells, finding that they did not alter cell survival in normoxic or anoxic conditions, when viability was already high. However, survival significantly increased in these cells after glucose and oxygen deprivation, indicating a protective role for the proteins.
Because ROS production is thought to be a key event in neuronal cell death after glucose deprivation and reoxygenation, and neuroglobin and cytoglobin are potential ROS scavengers, the group then looked at H2O2 levels. H2O2 did not increase in anoxic or glucose deprivation conditions in unmanipulated cells. Neuroglobin and cytoglobin overexpression both decreased H2O2 release compared to normoxic cells, while under-expressing the heme proteins increased H2O2levels, demonstrating that both neuroglobin and cytoglobin expression are correlated with oxidative stress, which may then result in cell death.
Although both the heme proteins decreased ROS release and increased cell survival, experiments looking at their normal expression suggested that neuroglobin and cytoglobin may have different functions, or at least different mechanisms of regulation. Neuroglobin mRNA and protein are upregulated under glucose deprivation and remain high upon reoxygenation. By contrast,cytoglobin is upregulated in anoxia and returns to basal levels upon reoxygenation. These increases are apparently not sufficient in vivofor neuroprotection, as alterations in ROS production and cell survival were seen only after heme protein over-expression. If antisense treatment for neuroglobin or cytoglobin increases ROS and cell death, then it is clear that upregulating these heme proteins makes good sense!
