- Top of page
- Materials and methods
Several mechanisms are thought to be involved in the progressive decline in neurons of the substantia nigra pars compacta (SNpc) that leads to Parkinson's disease (PD). Neurotoxin 6-hydroxydopamine (6-OHDA), which induces parkinsonian symptoms in experimental animals, is thought to be formed endogenously in patients with PD through dopamine (DA) oxidation and may cause dopaminergic cell death via a free radical mechanism. We therefore investigated protection against 6-OHDA by inhibiting oxidative stress using a gene transfer strategy. We overexpressed the antioxidative Cu/Zn-superoxide dismutase (SOD1) enzyme in primary culture dopaminergic cells by infection with an adenovirus carrying the human SOD1 gene (Ad-hSOD1). Survival of the dopaminergic cells exposed to 6-OHDA was 50% higher among the SOD1-producing cells than the cells infected with control adenoviruses. In contrast, no significant increased survival of (6-OHDA)-treated dopaminergic cells was observed when they were infected with an adenovirus expressing the H2O2-scavenging glutathione peroxidase (GPx) enzyme. These results underline the major contribution of superoxide in the dopaminergic cell death process induced by 6-OHDA in primary cultures. Overall, this study demonstrates that the survival of the dopaminergic neurons can be highly increased by the adenoviral gene transfer of SOD1. An antioxidant gene transfer strategy using viral vectors expressing SOD1 is therefore potentially beneficial for protecting dopaminergic neurons in PD.
Parkinson's disease (PD) is a neurological disorder characterized by a progressive and massive degeneration of dopaminergic cells in the substantia nigra pars compacta (SNpc). The resulting dopamine (DA) depletion in the striatum leads to the deterioration of motor function which manifests itself by tremor, bradykinesia, and rigidity. There is evidence that oxidative stress contributes to the nigral dopaminergic neuronal degeneration. This stress results from both increased generation of free radicals and impairment of the cell defence system for scavenging them (Dunnett and Björklund 1999). First, the nonenzymatic auto-oxidation of DA or its oxidation by monoamine oxidase-B would generate cytotoxic reactive oxygen species like hydrogen peroxide (H2O2) and superoxide (Olanow 1993). Activation of N-methyl-d-aspartate (NMDA) receptors by glutamate released from the neocortical or subthalamic inputs to the SN could further increase the generation of toxic free radicals. This process could be enhanced by the high levels of iron associated with low levels of ferritin in the SN of PD patients (Dexter et al. 1989, 1990) yielding cytotoxic hydroxyl radicals through the Fenton reaction (Youdim et al. 1993). In addition, the SN of PD patients have anormally low levels of reduced glutathione (GSH) (Perry et al. 1981) and of antioxidative enzyme activities (Ambani et al. 1975; Kish et al. 1985; Youdim et al. 1993), as well as abnormally high levels of lipid peroxides (Dexter et al. 1989).
The 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal dopaminergic system is commonly used as a model for PD. The neurotoxin is taken up by the dopaminergic neurons leading to the generation of neurotoxic reactive oxygen species resulting from DA auto-oxidation (Kumar et al. 1995). 6-OHDA has been detected in the urine of parkinsonian patients (Andrew et al. 1993), and it has been suggested to act endogenously in PD (Glinka et al. 1997). Injections of the neurotoxin into the rat striatum induce a reduction in the levels of GSH, GSH peroxidase (GPx), and SOD1, together with an increase in lipid peroxidation (Perumal et al. 1989; Kumar et al. 1995), a situation that mimics some aspects of the oxidative stress associated with PD. Antioxidants such as N-acetylcysteine, Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), or the C3 carboxyfullerene derivative, protect against 6-OHDA-mediated toxicity in dopaminergic neurons in vitro (Choi et al. 1999; Lotharius et al. 1999). Moreover, the protective potential of the SOD1 antioxidative enzyme against (6-OHDA)-induced toxicity has been demonstrated both in vitro using human SY5Y neuroblastoma cells (Tiffany-Castiglioni et al. 1982) and in vivo with SOD1 transgenic mice. In this study, the mice were protected against the neurotoxic effects of intracerebroventricular injection of the toxin (Asanuma et al. 1998). These studies all implicate free radicals in the (6-OHDA)-induced neurotoxicity.
One way to increase the antioxidant potential of dopaminergic cells both in vitro and in vivo is the transfer of genes encoding antioxidative enzymes into specific cell compartments, such that there is long-term overexpression of the corresponding proteins. This ‘antioxidant’ strategy of neuroprotection based on gene transfer technology should help increase the resistance of dopaminergic neurons to the oxidative stress generated during PD. We previously described the efficacy of this methodology for increasing survival of neuronal cells in which death was induced by exposure to glutamate or to β-amyloid in vitro (Barkats et al. 1996, 2000), and to the stress mediated by the intracerebral grafting procedure (Barkats et al. 1997). To analyse the protective potential of the DA neuronal gene transfer of SOD1 against (6-OHDA)-induced neurotoxicity, rat embryonic mesencephalic neurons were transduced with an adenovirus vector encoding the human SOD1 enzyme (Ad-SOD1) prior to exposure to 6-OHDA. Active human SOD1 was expressed in the (Ad-SOD1)-infected dopaminergic neurons. These neurons were significantly more resistant to 6-OHDA than either non-infected or control adenovirus-infected neurons. In contrast, the adenovirally mediated overproduction of GPx in dopaminergic neurons did not protect them against the toxic effects of 6-OHDA.
- Top of page
- Materials and methods
6-OHDA, which is commonly used to induce PD in experimental animals, is thought to cause dopaminergic cell death via a free radical mechanism. Using primary cultures of rat ventral mesencephalon, we examined the effect of SOD1 overexpression on the survival of dopaminergic neurons exposed to 6-OHDA. To overexpress the antioxidative enzyme in neurons, we used a gene delivery method based on recombinant adenoviruses. These viral vectors are among the most efficient for transducing postmitotic cells like neurons both in vitro and in vivo (Akli et al. 1993; Bajocchi et al. 1993; Davidson et al. 1993; Le Gal La Salle et al. 1993).
Co-labelling experiments combining Xgal and TH stainings in (Ad-βgal)-infected cultures revealed the transgene expression within both dopaminergic and non-dopaminergic cells. The percentage of dopaminergic neurons that were transduced after exposure to 25 MOI of Ad-βgal went beyond 50%. Production of the human SOD1 protein was evidenced in the mesencephalic cultures using a semiquantitative enzymatic assay and immunocytochemistry specific for the recombinant protein. Although we did not determine the exact number of the (Ad-SOD1)-transduced TH cells, it might be comparable to that of colabelled Xgal/TH cells (approximately 50%) since similar promoters (RSV) were used in both Ad-βgal and Ad-SOD1 constructions. The semiquantitative NBT assay showed that the intracellular SOD enzymatic activity in extracts of mesencephalic cells infected with 25 MOI Ad-SOD1 was nearly double that in uninfected cell extracts. This result suggests that cell infection with Ad-SOD1 led to a more than two-fold increase in cytoplasmic antioxidative activity (since only approximately 50% of dopaminergic neurons could be transduced with 25 MOI of adenoviral vectors).
Infection of mesencephalic cells with Ad-SOD1 prevented the death of a significant number of dopaminergic neurons exposed to 6-OHDA. The percent survival of TH+ neurons in cultures infected with Ad-SOD1 was significantly increased compared to controls treated with Adβgal or empty vectors (approximately two-fold). The difference with non-infected cells was less pronounced probably due to a residual toxicity of the adenoviral vector. The SOD1 enzyme is not secreted, and is exclusively synthesized in the cytoplasmic cell compartment. This suggests that only the (Ad-SOD1)-infected dopaminergic cells could be protected from (6-OHDA)-induced neurotoxicity. As only half the TH+ cells were transduced by adenoviral vectors, our results suggest that a large majority of the (Ad-SOD1)-transduced dopaminergic neurons were protected from 6-OHDA.
Potential trophic effects mediated by glia were insignificant in our experiments since as less than 1% of the cells in culture (under serum-free conditions) were glial cells (Frodl et al. 1994). A non-specific protective effect of treatment with adenoviral vectors, conceivably induced by a stress adaptative response of the cells, can also be excluded since no protective reaction against 6-OHDA toxicity was detected with control adenoviruses (empty vectors or vectors expressing β-galactosidase). Therefore, our results strongly suggest that the intraneuronal overexpression of SOD1 can inhibit dopaminergic cell death in (Ad-SOD1)-infected cultures.
The mechanisms underlying prevention of dopaminergic cell death by SOD1 overexpression are related to the sensitivity of dopaminergic neurons to (6-OHDA)-mediated oxidative stress. Earlier in vivo studies show that 6-OHDA injected into the striatum of rodents is transported into dopaminergic neurons where it is oxidized into reactive oxygen species (Kumar et al. 1995). Superoxide generated by 6-OHDA (Heikkila and Cohen 1973) can further cause dopaminergic cell death by interacting with nitric oxide to form the highly reactive peroxynitrite radical, or it can react with iron or copper to generate hydroxyl radicals. SOD, which detoxifies free radicals, may have a protective effect on dopaminergic neurons by scavenging (6-OHDA)-generated superoxide. Indeed, when overexpressed in transgenic animals, the superoxide-scavenging SOD1 protein was reported to the prevent the death of dopaminergic neurons induced by intracerebroventricular injection of 6-OHDA (Asanuma et al. 1998).
It has also been suggested that the neurotoxin 6-OHDA exerts its toxic effects on dopaminergic cells by production of H2O2 (Perumal et al. 1989). However, we did not find any significant effect of the overexpression of the H2O2-scavenging enzyme GPx in cultures infected with Ad-GPx before exposure to 6-OHDA. The lack of neuroprotective effect of GPx was unexpected because a partial protection of dopaminergic neurons against (6-OHDA)-induced neurotoxicity has been described in transgenic mice overexpressing GPx (Bensadoun et al. 1998). The discrepancy may result from a decreased availability of reduced glutathione (GSH) in our primary culture model. Indeed, the level of oxidized glutathione (GSSG) increases substantially after treatment with 6-OHDA (Spina et al. 1992). The concomitant decrease of GSH (Perumal et al. 1989) and/or glutathione reductase (GR) may therefore have limited the neuroprotective action of GPx in our experiments. This idea was also discussed by Bensadoun et al. (1998). The question of whether detoxification of H2O2 may contribute to reducing (6-OHDA)-mediated neurotoxicity could therefore be investigated by cotransducing cells with viral vectors encoding both GPx and GR, or GPx and the brain-derived neurotrophic factor (BDNF) which is known to induce the increase of GR activity (Spina et al. 1992).
In a previous study using ex vivo gene transfer technology, we showed that overexpression of SOD1 in dopaminergic neurons prior intrastriatal grafts in hemiparkinsonian rats increased the functional recovery of the animals (Barkats et al. 1997). This study suggested that free radicals were important agents in the dopaminergic cell death induced by the grafting procedure. Here, we further demonstrate the great neuroprotective potential of recombinant SOD1 adenoviral vectors for dopaminergic neurons in culture exposed to the 6-OHDA neurotoxin. Since it is clear that free radical homeostasis is extremely important for the survival of dopaminergic neurons, antioxidant gene transfer based on viral vector methodologies similar to those used in our study allows direct in vivo application of neuroprotective strategies in experimental models of neurodegenerative diseases. Scavenging enzymes, like SOD1, delivered directly to degenerating dopaminergic neurons by means of viral vectors, have enormous potential as neuroprotective agents in these experimental diseases. These antioxidant-based gene transfer strategies may reveal themselves more efficient than those using growth factors such as the glial cell line-derived neurotrophic factor (GDNF) or the BDNF. Indeed, a carboxyfullerene derivative (the C3 potent antioxidant) has recently been reported to outperform the highly protective effect of GDNF in the 6-OHDA model (Lotharius et al. 1999). Moreover, both GDNF and BDNF were recently suggested to act on dopaminergic neuron survival by activating the antioxidative enzyme systems (Spina et al. 1992; Chao and Lee 1999).
In conclusion, our study demonstrates the potential of an adenoviral vector encoding the human SOD1 to prevent (6-OHDA)-induced dopaminergic cell death by increasing the antioxidant potential of neurons. As 6-OHDA could act as an endogenous neurotoxin in PD, and because there is evidence for the contribution of oxidative stress in this disease, these results may be of significance for gene therapy of PD.