Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-D-aspartate (300 M, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nM) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the Nmethyl-D-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca 2؉ -activated neutral protease calpain I was readily detectable after the exposure to N-methyl-D-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-D-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.

An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 microm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochrome c, and a secondary rise in superoxide production 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activation, and cell death in control and rho(-) cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochrome c release occurs upstream of this defect.

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The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O 2 , whereas exposing these cells to 21% oxygen prohibited long-term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain-derived precursors cultured in 3 versus 21% oxygen after 1-2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self-renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons. Keywords: microarray, neural precursors, neurospheres, oxygen, proliferation, telomerase. Stem cells are undifferentiated cells that retain the ability to divide and proliferate throughout postnatal life to provide precursor cells that can differentiate into specialized cells. Neural stem cells can give rise to all the cells of the CNS including glial and neuronal cells (Gage 2003). As a result of their ability to differentiate following transplantation, these cells represent a potentially valuable therapeutic tool for the treatment of genetic, traumatic, inflammatory or degenerative neurological disorders (Studer et al. 1998;Storch and Schwarz 2002;Conti et al. 2003). To enable long-term expansion, all precursor cells must maintain the capacity to self-renew and a positive balance of proliferation versus differentiation or cell death. Therefore, it is crucial to understand the mechanisms governing cell division, differentiation and senescence (Sharpless and DePinho 2004).Mammals have evolved complex circulatory systems to ensure that every cell receives sufficient oxygen for normal metabolic processes. Lack of oxygen is defined as a reduction in oxygen supply to the tissues to below physiological levels. Hypoxia can develop as a result of ischaemia, representing a pathological component of stroke and heart attack. In addition to being a consequence of the growth of a malignant tumour and so a potential marker, hypoxia also acts to promote tumour development (Brizel et al. 1996;Brat et al. 2004). Address correspondence and reprint requests to Javorina Milosevic, Department of Neurology, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany. E-mail: javorina.Milosevic@medizin.uni-leipzig.deAbbreviations used: ADA, adenosine deaminase; bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2-deoxyuridine; DA, dopamine; DAPI, 6¢-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF...

Exposure of rat hippocampal neurons or human D283 medulloblastoma cells to the apoptosis-inducing kinase inhibitor staurosporine induced rapid cytochrome c release from mitochondria and activation of the executioner caspase-3. Measurements of cellular tetramethylrhodamine ethyl ester fluorescence and subsequent simulation of fluorescence changes based on Nernst calculations of fluorescence in the extracellular, cytoplasmic, and mitochondrial compartments revealed that the release of cytochrome c was preceded by mitochondrial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-xL, but not pharmacological blockade of outward potassium currents, inhibited staurosporine-induced hyperpolarization and apoptosis. Dissipation of mitochondrial potassium and proton gradients by valinomycin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine-induced hyperpolarization, cytochrome c release, and caspase activation. This effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se also caused release of cytochrome c from mitochondria. In contrast to staurosporine, however, valinomycin-induced cytochrome c release and cell death were not associated with caspase-3 activation and insensitive to Bcl-xL overexpression. Our data suggest two distinct mechanisms for mitochondrial cytochrome c release: (1) active cytochrome c release associated with early mitochondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.