Invited Symposium: Quinones and Other Reactive Oxygen Species in Neurobiologic, Apoptotic, and Neurotoxic Processes
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Introduction
Parkinson's disease is a neurodegenerative disorder that affects dopaminergic systems in the human brain (Mochizuki et al., 1996; Ziv et al., 1997). Several models have been used to investigate cellular and molecular mechanisms that might be involved in the causation of PD (Cadet and Brannock, 1998). These include, among others, the use of 6-hydroxydopamine (6-OHDA) (Asanuma et al., 1998; Michel and Hefti, 1990), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Itzhak et al., 1998), and of dopamine (DA) itself (Cheng et al., 1996; Hoyt et al., 1997; Often et al., 1997). The neurodegenerative effects of these compounds are thought to involve the generation of free radicals (FR) (Berman et al., 1996; Cadet and Brannock, 1998; Hastings and Zigmond, 1997). In the case of DA, the production of FR is thought to occur via oxidation or metabolic breakdown by monoamine oxidase to produce reactive oxygen species (ROS) such as superoxide radicals, hydrogen peroxide and hydroxyl radicals (Cadet and Bannock, 1998). The idea is supported by the recent evidence that the toxic effects of the DA metabolite, 6-OHDA, involve the production of superoxide radicals since transgenic mice that overexpress human CuZn superoxide dismutase are protected against the neurodegenerative effects of this compound on nigrostriatal dopaminergic neurons (Asunama et al., 1998).
While many of these studies have documented a role for free radicals in models of PD (Cadet and Brannock, 1998), it is, however, not clear why some people develop PD whereas other individuals do not since DA is presumably being metabolized in a similar fashion among various individuals and should be producing free radicals in the substantia nigra pars compacta (SNpc) of everybody. This argument, thus, raises the possibility that dysregulation of prodeath and antideath mechanisms in PD patients might play an essential role in the development of the disorder. One of the potential sources of dysfunction is in the proto-oncogene, bcl-2. Bcl-2 was first identified at the chromosomal breakpoint (14, 18) in B cell lymphoma (Tsujimoto et al., 1984). Subsequent studies have shown that bcl-2 can act to promote cell survival, to prevent damage caused by oxidative stress, and to block apoptic cell death (Cadet et al., 1998; Chou et al., 1994; Hockenbery et al, 1993; Kane et al., 1993; Nunez et al., 1990). In addition, bcl-2 overexpressing transgenic mice are protected against developmental cell death (Martinou et al, 1994) whereas bcl-2 knockout mice are more susceptible to certain insults (Hockman et al.,1998).
Because bcl-2 acts as an antideath agent, it was of interest to determine if bcl-2 overexpression could protect against cell death caused by DA. Herein, we have shown that bcl-2 can indeed block apoptosis caused by DA in vitro. These results provide partial support for the idea that dysregulation of bcl-2 related gene products might be involved in neurodegeneration of nigrostriatal DA pathways.
Materials and Methods
Cell Culture and Treatment of the Cultures and Assessment of Toxicity The bcl-2 expressing cells have been shown to be resistant to the toxic effects of serum withdrawal, glucose deprivation, and membrane peroxidation (Zhong et al., 1993) as well as methamphetamine-induced apoptosis (Cadet et al., 1997).
Cytotoxicity was measured by the release of the lactate dehydrogenase enzyme into the culture supernatant upon damage of the plasma membrane or during cell death. An aliquot of culture supernatant was collected cell free and incubated with the reaction mixture from a Kit (Cytotoxicity detection, Boehringer).
Analysis of Apoptosis
Confocal Microscopy: Acetone-fixed slides mounted cells were used. They were stained with acridine orange for 15 minutes using a final concentration of 10 ug/ml. After staining, the slides were washed with running deionized water, air dried and mounted with slow-fade mounting solution (Molecular Probes). Cells were imaged using a Zeiss confocal LSM 410. Acridine orange was excited at 488 nM and the emission was measured with fluorescein filter.
Flow Cytometry: Apoptosis was assessed by flow cytometry using APO-BRDU Kit (Phoenix Flow Systems Inc., San Diego CA) as described in the manufacturer=s protocol. This Kit is a two color staining method that labels DNA strand breaks and total cellular DNA; this approach serves to detect cells undergoing apoptosis using flow cytometry (Li and Darzyn-kiewicz, 1995). 3'-Hydroxyl ends of fragmented DNA are labeled with bromolated deoxyuridine triphosphate nucleotides (BrdUTP) using deoxyl-nucleotidyl transferase. These sites are then identified by a fluorescent labeled anti-BrdU monoclonal antibody. Non-apoptotic cells do not incorporate Br-dUTP owing to the lack of exposed 3'-hydroxyl DNA ends. ROS analysis Peroxide levels were measured by using the dye 2,7-dichlorofluorescin (DCF). DCF fluorescence was measured over a period of 3 h using a Cytofluor 4000 plate reader set at EX=485 and Em=530. We compared changes in ROS production with and without addition of dopamine.
Results
Dopamine caused dose-dependent increases in LDH release, with 40 uM causing death of almost all of the cells. Overexpression of bcl-2 caused marked reduction of DA-induced cell death which plateaued at around 40% of total LDH released.
Microscopic analysis of DA-induced effects revealed that DA exposure caused the cytoplasm to shrink. In addition, the nuclei were fragmented in small pieces. As in the case of LDH release, over-expression of bcl-2 significantly attenuated these DA-induced nuclear fragmentation.
Further evidence that DA-induced cell death is due to apoptotic was provided by flow cytometric measurements of internucleosomal breakdown of DNA. DA caused dose-dependent DNA breaks. Bcl-2 overexpression caused significant protection against the DA-induced apoptotic changes at all doses of DA used in the present study.
Because it has been suggested that DA exerts its toxic effects via the production of reactive oxygen species (Cadet and Brannock, 1998) and because bcl-2, in addition to its other effects, is also thought to behave as an antioxidant (Kane et al., 1993), we also tested the possibility that DA could cause ROS production in this cell line. In addition, we assessed if bcl-2 could reduce DA-induced ROS production. DA also caused marked increases in ROS during the 2-hr of observation. Bcl-2 overexpression caused a marked reduction in ROS at all time points examined.
Discussion and Conclusion
The main findings in those experiments are: (1) DA caused apoptosis in immortalized neural cell line, (2) DA caused marked increases in ROS production, and (3) bcl-2 significantly attenuated the toxic effects of DA by reducing ROS production. The demonstration that DA can cause apoptosis is in accord with a number of recent observations using primary cultures and cell lines including PC 12 cells (Often et al., 1997; Shirvan et al., 1997). Our present results are also compatible with the report that DA-induced apoptosis can be exacerbated by a bcl-2 antisense oligo (Masserano et al., 1996).
The findings that DA exposure caused a marked increase in ROS production is also consistent with the idea that catecholamines including DA and 6-OHDA might produce their toxicity via ROS production (Cadet and Brannock, 1998). Recent experiments have suggested that superoxide radicals, hydrogen peroxide, and hydroxyl radicals might participate in a toxic cascade to destroy both striatal DA terminals and nigral cell bodies (see review by Cadet and Bannock, 1998). The demonstration that bcl-2 can cause a marked reduction in ROS production supports the view that bcl-2 can indeed work as an antioxidant (Cadet et al., 1997; Kane et al.; 1993) in addition to its other cellular and molecular effects (Akao et al., 1994). These results also point to the possibility that the bcl-2-protective pathway might be one site of possible dysregulation that needs to be investigated as a possible cause of Parkinsonism. Because bcl-2 is localized in organelles such as the mitochondria (Akao et al.., 1994) that are active sources of ROS, it is indeed not far-fetched to envision that bcl-2 itself or one the bcl-2-related protectants could be dysfunctional in Parkinson=s disease. This remains to be demonstrated.
In summary, the data presented provide further support for the idea that PD might be secondary to direct toxic effects of DA itself. Because DA should be toxic in all human beings and because not everybody develops PD, a dysfunctional antioxidant system might participate in the development of PD. The present observations that bcl-2 can attenuate the toxic effects of DA and the previous report that an antisence bcl-2 oligo can exacerbate DA-induced cell death (Masserano et al., 1996) suggest that down-regulation of bcl-2 in PD might be an enhancer of the toxic effects of DA in PD patients. While this idea is supported by these in vitro studies, it has yet to be fully investigated by in vivo studies. Finally, investigations of bcl-2 systems and other cell-death related genes and their interactions with brain DA systems might help in the elucidation of cellular and molecular mechanisms involved in nigral cell death observed in Parkinson's disease.
References
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