Abstract:We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular Kconcentration ([K~] 0),a significant part of the genomic DNA was already cleaved to highmolecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K~]0 deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclearfragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning .-~4 h after the removal of high [K~]0and developed to nuclear condensation 4 h later. Six hours after high [K~]0 deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K~, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K~]0. Similar to high [K~]0 deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K~]0 removal. An~-~40% decrease in RNA and protein synthesis was detected by 6 h of [K~]0removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activityevents that occurred within the critical time-may indicate that these processes have an important part in the mechanism that committed the neurons to die.

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis. E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -CysPro-Tyr-Cys-active site, penetrates into cerebellar granule neurons and exerts its activity via NF-B activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although The thiol-disulfide metabolism and balance contributes to the maintenance of the cellular redox (reduction/oxidation) state. Thiol redox control (1) can affect a protein's synthesis and folding, the assembly into multimeric complexes, the enzymatic activity or the binding activity of transcription factors (Refs. 2 and 3; for review, see Ref. 4). Intracellular redox-active molecules such as thioredoxin, glutaredoxin, and Ref-1, and low molecular weight thiols such as glutathione (GSH), distribute and maintain a reduced cytosolic environment in the normal cell.Dopamine (DA), 1 the endogenous neurotransmitter of the nigro-striatal pathway, is a powerful oxidant that exerts its toxic potential through its oxidative metabolites. Administration of DA into the rat striatum (5) caused pre-and postsynaptic damage. Intraventricular injection of DA into rats resulted in dose-dependent death of the animals (6). In vitro studies have shown that DA can cause cell death in mesencephalic, striatal, and cortical primary neuron cultures (7-11). Moreover, we have shown that DA-induced cell death in sympathetic, cerebellar granule neurons, PC-12 cells, and thymocytes has all the characteristic features of apoptotic cell death (12)(13)(14).

Excessive production of reactive oxygen species in living cells may damage their biological components. This condition, referred to as oxidative stress, is a common denominator of pathological conditions. Cells have evolved a wide array of antioxidant mechanisms including small reducing molecules (e.g. glutathione, ascorbic acid), antioxidative enzymes (e.g. catalase, superoxide dismutase, glutathione peroxidase; for review, see Refs. 1-4), and oxidoreductase enzymes such as thioredoxin and glutaredoxin (Grx) 1 (5).Grx are antioxidant enzymes by virtue of the reducing power of their active site (CXXC), which catalyzes the transfer of electrons from reduced glutathione to disulfides (5). This thiol disulfide interchange reaction is crucial for the maintenance of intracellular redox homeostasis, especially under oxidative stress (6). Mammalian Grx is widely expressed in different cell types, including neurons (7-11). The enzyme can restore the activity of glutathionylated proteins containing mixed disulfides between a protein thiol and GSH (inactive form) by reducing the disulfide bridge to give reduced GSH and the active protein form containing a free thiol. Examples of such GSHthiol regulation of activity can be found in tyrosine phosphatase 1B (13), phosphofructokinase (14, 15), nuclear factor-I (16), and polyomavirus enhancer-binding protein 2 (17). Thanks to the antioxidant properties of Grx activity, human Grx and Escherichia coli Grx2 can rescue cerebellar granule neurons from dopamine (DA)-induced oxidative stress (18).DA, the endogenous neurotransmitter of the nigrostriatal pathway, is a powerful oxidant that exerts its toxic effects through its oxidative metabolites. DA-induced oxidations are generally implicated in neurodegenerative processes (19,20) especially in Parkinson's disease (21,22). Administration of DA to rat striatum caused pre-and postsynaptic damage (23). Intraventricular injection of DA in rats resulted in dose-dependent death of the animals (24). In vitro studies have shown that DA can cause cell death in mesencephalic, striatal, and cortical primary neuron cultures (25-29). DA-induced cell death in sympathetic, cerebellar granule neurons, PC-12 cells, and thymocytes has all the features of apoptotic cell death (30 -32). Apart from the administration of Grx, the toxic effects of DA can be prevented by the application of small molecular weight antioxidants such as N-acetylcysteine, catalase, ascorbic acid, and dithiothreitol (30,31,33,34).Little is known about the molecules and signaling pathways involved in DA-induced apoptosis. Enhancement of the DNA binding activity of NF-B protects neurons from DA-induced apoptosis (18). A survival signal pathway that might activate NF-B is the Ras/PI3K/Akt/NF-B cascade (35,36).Ras is a family of proteins involved in the regulation of cell proliferation, cytoskeletal rearrangements, and differentiation and survival of different cell types (37,38). Ras activation is localized on the inner surface of the cell membrane where it * This research was suppor...

1. The pathogenesis of the selective degeneration of the dopaminergic neurons in Parkinson's disease is still enigmatic. Recently we have shown that dopamine can induce apoptosis in postmitotic neuronal cells, as well as in other cellular systems, thus suggesting a role for this endogenous neurotransmitter and associated oxidative stress in the neuronal death process. 2. Dopamine has been shown to be capable of inducing DNA damage through its oxidative metabolites. p53 is a transcription factor that has a major role in determining cell fate in response to DNA damage. We therefore examined the possible correlation between dopamine-triggered apoptosis, DNA damage and levels of total phosphorylated p53 protein in cultured mouse cerebellar granule neurons. 3. Marked DNA damage and apoptotic nuclear condensation and fragmentation were detected within several hours of exposure to dopamine. An associated marked threefold increase in p53 phosphorylation was observed within this time window. Using a temperature-sensitive p53 activation system in leukemia LTR6 cells, were found that p53 inactivation dramatically attenuated dopamine toxicity. 4. We therefore conclude that DNA damage and p53 activation may have a role in mediating dopamine-induced apoptosis. Modulation of the p53 system may therefore have a protective role against the toxicity of this endogenous neurotransmitter and associated oxidative stress.