Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H(2)O(2)-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (γ- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H 2 O 2 . Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.

Neuromelanin in the substantia nigra may be associated with the pathogenesis of nigral cell death in Parkinson's disease. We used synthetic dopamine-melanin (DA-M) as a model compound for neuromelanin and examined its toxic effects on mice cerebellar granule cells and a rat pheochromocytoma cell line (PC12). The DA-M and dopamine-treated cells showed an accumulation of black deposits when examined by light microscopy. Electron microscopy revealed different stages of DA-M phagocytosis, starting with DA-M binding, engulfment of the particles and the formation of phagosomes located in the cytoplasm. Using absorption assays, we found that NaN3 and low temperature inhibit the internalization of DA-M, pointing to an energy-dependent phagocytosis mechanism. These results suggest that neuromelanin can be phagocytised by neuronal cells which may thus be subjected to its toxic effects. These findings may contribute to our understanding of the formation and disposition of neuromelanin and its possible role in the etiology of Parkinson's disease.

Using in situ hybridization, the presence of T-cell lymphotrophic virus type I (HTLV-I) was shown in blood lymphocytes of one tropical spastic paraparesis (TSP/HAM) patient and in two asymptomatic carriers. HTLV-I was also detected in epithelial cells derived from mouthwash of the TSP/HAM patient. Mouthwash of one of the carriers showed an infected lymphocyte while mouthwash of the other carrier was negative. The infected epithelial cells stained both in the nucleus and in the cytoplasm, which indicated the presence of the virus in both subcellular compartments. Our observations suggest that saliva cells, lymphocytes and epithelial cells, may potentially participate in oral transmission of HTLV-I.

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-hbcl-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP+. Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.

Abstract: Bcl‐2 is an antiapoptotic protein located in the outer mitochondrial membrane. Cellular perturbations associated with programmed cell death may be the consequence of disrupted mitochondrial function as well as excessive production of reactive oxygen species (ROS). Numerous studies indicate that Bcl‐2 is involved in opposing cell death induced by oxidative stimuli, but its mode of action is uncertain. We reexamined the role of Bcl‐2 by using a loss‐of‐function model, Bcl‐2 knockout mice. Brains from Bcl‐2‐deficient mice had a 43% higher content of oxidized proteins and 27% lower number of cells in the cerebellum relative to wild‐type mice. Incubation of cerebellar neurons from Bcl‐2 +/+ brains with 0.5 mM dopamine caused 25% cell death, whereas in Bcl‐2‐deficient cells, it resulted in 52% death; glial cells provided protection in both cultures. Splenocytes from Bcl‐2‐deficient mice were also killed more effectively by dopamine as well as paraquat. Bcl‐2‐deficient mice did not survive intraperitoneal injection of MPTP, which caused a decrease in dopamine level in the striatum of Bcl‐2 +/− brains, which was more significant than in wild‐type mice. When compared with Bcl‐2 +/+ brains, brains of 8‐day‐old Bcl‐2‐deficient mice had higher activities of the antioxidant enzymes GSH reductase (192%) and GSH transferase (142%), whereas at the age of 30 days, GSH peroxidase was significantly lower (66%). Activities of GSH transferase and GSH reductase increased significantly (158 and 262%, respectively) from day 8 to day 30 in Bcl‐2 +/+ mice, whereas GSH peroxidase decreased (31%) significantly in Bcl‐2−/− animals. In summary, our results demonstrated enhanced oxidative stress and susceptibility to oxidants as well as altered levels of antioxidant enzymes in brains of Bcl‐2‐deficient mice. It is concluded that Bcl‐2 affects cellular levels of ROS, which may be due to an effect either on their production or on antioxidant pathways.

We have recently shown that dopamine (DA) can trigger apoptosis, an active program of cellular self-destruction, in various neuronal cultures and proposed that inappropriate activation of apoptosis by DA and or its oxidation products may initiate nigral cell loss in Parkinson's disease (PD). Since DA toxicity may be mediated via generation of oxygen-free radical species, we examined whether DA-induced cell death in PC12 cells may be inhibited by antioxidants. We have found that the thiol containing compounds, reduced glutathione (GSH),N-acetyl-cysteine (NAC), and dithiothreitol (DTT) were markedly protective, while vitamins C and E had lesser or no effect. The thiol antioxidants and vitamin C but not vitamin E, prevented dopamine autooxidation and production of dopamine-melanin. Their protective effect has also manifested by inhibiting DA-induced apoptosis; DNA fragmentation was prevented as was shown histochemically by thein situend-labeled DNA technique (TUNEL). Intracellular GSH and other thiols constitute an important natural defense against oxidative stress. We have found that depletion of cellular GSH by the addition of phoron, a substrate of glutathione transferase, and buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl transpeptidase, significantly enhanced DA toxicity. Cotreatment with NAC rescued the cells from the toxic effect of BSO + DA, and phoron + DA, while addition of GSH provided only partial protection from BSO + DA toxicity. Our data indicate that the thiol family of antioxidants, but not vitamins C and E, are highly effective in rescuing cells from DA-induced apoptosis. Further study of the mechanisms underlying the unique protective capacity of thiol antioxidants may lead to the development of new neuroprotective therapeutic strategies for PD.

The function of neuromelanin (NM), the oxidized dopamine (DA) polymer, within the DA-producing cells in the human and primate substantia nigra (SN), is still an enigma. Some studies show that the vulnerability of nigral neurons in Parkinson's disease is correlated to their toxic NM content, while others suggest that it contributes to cellular protection. We showed recently that DA, the endogenous nigral neurotransmitter, triggers apoptosis, an active program of cellular self-destruction, in neuronal cultures. In the present study, we exposed cells to synthetic dopamine–melanin (DA–M) and analysed the cellular and genetic changes. We found that exposure of PC12 cells to DA–M (0.5mg/ml for 24h) caused 50% cell death, as indicated by trypan blue exclusion assay and 3H-thymidine incorporation. Gel electrophoresis DNA analysis of PC12 cells treated with DA–M showed the typical apoptotic DNA ladder, indicating inter-nucleosomal DNA degradation. The DNA fragmentation also was visualized histochemically in situ by DNA end-labeling staining (the TUNEL method). The FeCl2 (0.05mM) significantly increased DA–M toxicity, while desferrioxamine, an iron chelator, totally abolished the additive toxicity of iron. The contribution of oxidative stress in this model of DA–M-induced cell death was examined using various antioxidants. In contrast to DA, inhibition of DA–M toxicity antioxidants by reduced glutathione (GSH), N-acetyl cysteine, catalase and Zn/Cu superoxide dismutase (SOD) was very limited. In conclusion, we found that DA–M may induce typical apoptotic death in PC12 cells. Our findings support a possible role of NM in the vulnerability of the dopaminergic neural degeneration in Parkinson's disease. The differential protective effect by antioxidants against toxicity of DA and DA–M may have implications for future neuroprotective therapeutic approaches for this common neurological disorder.

Exposure of mouse thymocytes to dopamine caused apoptosis (programmed cell death). This was manifested by cellular condensation and membrane damage shown by flow cytometry measurements and scanning electron microscopic study. Dopamine also affected thymocytic nuclei and their genomic DNA integrity. Most of the DNA molecules accumulated in a subdiploid peak in flow cytometry analysis, indicating DNA fragmentation to small particles. DNA analysis showed the typical pattern of 'DNA ladder' caused by internucleosomal DNA cleavage. X-ray microanalysis of the cellular elements of dopamine-treated cells showed elevation of sodium (Na), chloride (Cl) and calcium (Ca) peaks, accompanied by reduction in phosphate (P) concentrations. Comparison of the potassium (K) and P concentrations showed significant differences between the two major death processes: necrosis (induced by exposure to sodium azide (NaN3)) and apoptosis (induced by dopamine). High concentrations of K indicated cell viability while reductions in P and elevations in Ca levels were found to be typical of apoptotic cell death. The antioxidant dithiothreitol (DTT) suppressed dopamine-induced apoptosis in thymocytes, suggesting that its toxicity may be mediated via generation of reactive oxygen radicals. Our study suggests that under certain circumstances, dopamine and/or its metabolites, may induce a process of apoptotic cell death of the dopamine-producing cells in the substantia nigra. Increased accessibility of dopamine to the nigral cell nucleus or inability to scavenge excess free radicals generated from dopamine oxidation triggering programmed cell death, may cause the progressive nigral degeneration in Parkinson's disease.