David Blum

Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. Although the etiology of PD is unknown, major biochemical processes such as oxidative stress and mitochondrial inhibition are largely described. However, despite these findings, the actual therapeutics are essentially symptomatical and are not able to block the degenerative process. Recent histological studies performed on brains from PD patients suggest that nigral cell death could be apoptotic. However, since post-mortem studies do not allow precise determination of the sequence of events leading to this apoptotic cell death, the molecular pathways involved in this process have been essentially studied on experimental models reproducing the human disease. These latter are created by using neurotoxic compounds such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or dopamine (DA). Extensive study of these models have shown that they mimick, in vitro and in vivo, the histological and/or the biochemical characteristics of PD and thus help to define important cellular actors of cell death presumably critical for the nigral degeneration. This review reports recent data concerning the biochemical and molecular apoptotic mechanisms underlying the experimental models of PD and correlates them to the phenomena occurring in human disease.

p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells.

p53, Bax and Bcl-xL proteins have been implicated in apoptotic neuronal cell death. We have investigated whether those proteins are involved in 6-OHDA-induced PC12 cell death. After a 24-h exposure to the neurotoxin (100 microM), morphological evidence for apoptosis was observed in PC12 cells. Up-regulation of p53 and Bax proteins was demonstrated 4 and 6 h, respectively, after 6-OHDA treatment; in contrast, no change in Bcl-xL levels was found. These findings suggest that p53 and Bax could be relevant markers of neuronal apoptosis as previously described in kainic acid- or ischemia-induced neuronal cell death and may participate to neuronal degeneration in Parkinsons disease.

Extracellular toxicity of 6-hydroxydopamine on PC12 cells

6-hydroxydopamine (6-OHDA) is usually thought to cross cell membrane through dopamine uptake transporters, to inhibit mitochondrial respiration and to generate intracellular reactive oxygen species. In this study, we show that the anti-oxidants catalase, glutathione and N-acetyl-cysteine are able to reverse the toxic effects of 6-OHDA. These two latter compounds considerably slow down 6-OHDA oxidation in a cell free system suggesting a direct chemical interaction with the neurotoxin. Moreover, desipramine does not protect PC12 cells and 6-OHDA is also strongly toxic towards non-catecholaminergic C6 and NIH3T3 cells. These results thus suggest that 6-OHDA toxicity on PC12 cells mainly involves an extracellular process.

Unlike MPP+, apoptosis induced by 6-OHDA in PC12 cells is independent of mitochondrial inhibition

The mechanisms of 6-hydroxydopamine (6-OHDA) cytotoxicity were studied in vitro using the PC12 cell line. Following a 24 h exposure, this neurotoxin induced apoptosis and a dose-dependent decrease in cell survival. The presence of monoamine oxidase inhibitors, tranylcypromine and clorgyline, together with 6-OHDA had neither synergistic nor protective effects. Unlike 1-methyl-4-phenylpyridinium (MPP+), 6-OHDA toxicity to PC12 cells remained unchanged when glycolysis was prevented by either depleting glucose from the culture medium or growing the cells in low-glucose medium containing 2-deoxy-glucose. These results suggest that the inhibition of mitochondrial respiration is not responsible for the cell death induced by 6-OHDA.

6-Hydroxydopamine-induced nuclear factor-kappaB activation in PC12 cells

The involvement of nuclear Factor-kappa B (NF-kappa B) transcription factor in PC12 cell death triggered by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was investigated. Results show that oxidative stress generated by 6-OHDA activates NF-kappa B. When the NF-kappa B activation was inhibited by parthenolide, PC12 cell death induced by 6-OHDA was significantly increased, thus suggesting an involvement of this transcription factor in a protective mechanism against 6-OHDA toxicity. To further assess this hypothesis, we studied the involvement of NF-kappa B in the protective effect of two anti-apoptotic genes, bcl-2 and bfl-1. Although Bcl-2 and Bfl-1 expression normally protects PC12 cells from 6-OHDA, parthenolide strongly decreased the beneficial effects afforded by transgene expression. These results suggest: (1) that the transcription factor NF-kappa B is likely associated with the protection of catecholaminergic PC12 cells and (2) that the protective effects afforded by bcl-2 and bfl-1 expression may be dependent on NF-kappa activation.

Mycoplasmas as gene therapy vectors

Resolving contradictory reports on cell aging To the editor: In a recent commentary on cell aging in this journal (Nat. Biotechnol. 16:396, May 1998), I pointed out that human fibroblasts leave the division cycle irreversibly at an increasing rate with each passage in culture1 and concluded that the Hayflick limit represents the number of divisions of the longest surviving clone. I was recently informed of a paper published a decade earlier that demonstrated that monoclonality developed in mass cultures of lymphocytes from all humans tested, with no difference between the replicative lifespan of mass cultures from birth to old age2. It also showed, however, a significant inverse relation between donor age and replicative lifespan among randomly isolated individual lymphocyte clones. The replicative lifespan of the lymphocyte clones is much shorter than that of the mass cultures. The implication is that rare clones that have a prolonged replicative lifespan in mass culture define the Hayflick limit equally for young and old donors, but mask the evidence for differences in accumulated damage between the great majority of lymphocytes from different age groups. This analysis provides a plausible explanation for the recent failure to find an agerelated difference in replicative lifespan between fibroblasts from young and old healthy donors in the largest study ever done,3 in contrast to the weak relationship reported in the classic study on the subject4. The latter was based mainly on cells from refrigerated cadavers or autopsies3 in which there was likely to be postmortem death of cells that would tend to eliminate the rare, long-lived clones and leave the more representative ones to determine lifespan. The present analysis would also account for the failure of other investigators to find an age relationship for cell divisions in fibroblasts from healthy donors while finding it in diabetic and prediabetic patients5. The clonal results presumably reflect the accumulation of genetic damage in cells with age,6,7 but give no reason8,9 to believe there is a fixed limit to cell division in vivo, much less10 a mechanism such as telomere length to count divisions. Harry Rubin Department of Molecular and Cellular Biology University of California, Berkeley (hrubin@uclink2.berkeley.edu)

A cautionary note on the use of stable transformed cells

Gene transfection and ectopic expression is a widely used method in experimental biology. In the present report, we would like to point out that this approach may, in certain circumstances, lead to a modification of the transfected cell phenotype. Indeed, we observed that after transfection of bcl-2 gene in the neuronal PC12 cell line some of the selected clones have lost their neuronal and catecholaminergic characteristics, i.e. TH expression and ability to grow neurites in response to NGF. Thus, the resistance of some PC12-Bcl-2 clones against neurotoxic insults may not necessarily reflect the potential benefit afforded by Bcl-2 expression. We therefore encouraged authors to verify cell phenotype after stable transfection to avoid misinterpretation of their results.