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Mutations in α-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of α-synuclein overexpression on the response of cells to various insults. Wild-type α-synuclein and α-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type α-synuclein delayed cell death induced by serum withdrawal or H2O2, but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP+). By contrast, wild-type α-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type α-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant α-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant α-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal α-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.
Parkinson's disease (PD) results from degeneration of dopaminergic neurons in the substantia nigra (Jenner and Olanow 1998). Although most cases appear sporadic and of unknown cause, familial Parkinson's disease (FPD) can occur and defects in the gene encoding α-synuclein protein are sometimes involved (Polymeropoulos et al. 1997). α-synuclein is a 15-kDa protein containing 140 amino acids and its major expression site is the presynaptic terminal. The amino terminal region contains five consensus sequence repeats, ‘KTKEGV’ and the carboxyl terminal is rich in acidic amino acids (Jakes et al. 1994). α-Synuclein is present in Lewy bodies, which are a characteristic feature of PD, and of dementia with Lewy body disease (Spillantini et al. 1998a). Abnormal staining for α-synuclein is found in neurofilament inclusions in multisystem atrophy (Spillantini et al. 1998b) and in Alzheimer's disease (Goedert et al. 1998). Two mutated α-synuclein proteins (A30P and A53T) have been found in some cases of FPD (Polymeropoulos et al. 1997; Kruger et al. 1998). The mutant α-synucleins have been suggested to self-aggregate, and to aggregate with wild-type α-synuclein and other proteins, to give insoluble deposits that cannot undergo proteolysis by the proteasome (Mezey et al. 1998). Oxidative stress may also promote the aggregation of α-synuclein (Hashimoto et al. 1999a). The proteasome is important in clearing abnormal proteins, including oxidatively damaged proteins within the cells (Grune et al. 1995; Fenteany and Schreiber 1998).
In the present study, we investigated how cell death induced by a variety of insults was modulated by overexpression of wild-type or FPD-linked mutant α-synuclein proteins. Since oxidative stress is involved in PD (Jenner and Olanow 1998), we tested insults related to this process, namely H2O2 and 4-hydroxy-2-trans-nonenal (HNE). The latter is a toxic unsaturated aldehyde produced during lipid peroxidation and which is present in increased amounts in parkinsonian substantia nigra (Yoritaka et al. 1996). We also examined serum deprivation, the mitochondrial complex I inhibitor, MPP+ (Singer et al. 1987), the protein kinase inhibitor, staurosporine (Fujita-Yamaguchi and Kathuria 1998) and an inhibitor of proteasome function, lactacystin (Fenteany and Schreiber 1998). To ensure that our results were not unique to a particular cell line, we compared two different cell lines, namely NT-2/D1 (NT-2) and SK-N-MC.
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There is considerable evidence that oxidative stress is associated with the progression of sporadic PD and may be an early stage in its development (Sian et al. 1994; Beal 1997; Jenner and Olanow 1998). For example in substantia nigra from PD patients elevated levels of HNE, protein carbonyls and 8-OHG have been reported (Chirico 1994; Alam et al. 1997a, 1997b; Jenner and Olanow 1998). Defects in mitochondria, especially complex I, have also been reported and may themselves lead to further oxidative stress (Schapira 1999). There is growing evidence that apoptosis contributes to neuronal cell death in substantia nigra in PD (Stefani et al. 1997). Considerable excitement has been generated by reports that some cases of FPD are associated with mutations in α-synuclein (Mezey et al. 1998), but the mechanisms by which this leads to cell death are unknown. It is important to determine to what extent α-synuclein mutations affect the response of cells to injurious agents.
Over-expression of wild-type α-synuclein in two different cell lines did not affect growth (data not shown) or viability, and had little or no effect on most of baseline parameters of oxidative damage (8-OHG and lipid peroxidation) or on the levels of 3-nitrotyrosine, although protein carbonyls were significantly elevated. Protein carbonyls are regarded as a general assay of protein damage, generated by direct free radical attack upon, or the binding of certain aldehydes (including HNE) to, amino acid residues in proteins (Oliver et al. 1990).
We examined the effect of various toxic insults, relevant to PD, upon the viability of α-synuclein transfectants, including H2O2, MPP+ and serum withdrawal. H2O2 is generated in increased amounts by monoamine oxidase activity in parkinsonian substantia nigra (Cohen et al. 1997). MPP+ is a mitochondrial electron transport chain complex I inhibitor that models the complex I defect found in PD (Schapira 1999) and HNE is present in increased amounts in parkinsonian substantia nigra (Yoritaka et al. 1996). Serum deprivation is a widely used model for neurotrophic factor deprivation, and such factors are being widely investigated as neuroprotective agents (Bjorklund et al. 1997). Cells transfected with wild-type α-synuclein were less sensitive to cell death induced by H2O2 or serum withdrawal. Death occurred largely by apoptosis, as revealed by morphology examinations, PI, Hoechst 33258, TUNEL and DNA laddering. However, there was no effect of wild-type α-synuclein transfection on death induced by MPP+.
By contrast, both of the mutant α-synucleins examined were deleterious to both cell lines. They slowed growth rate even under normal culture conditions (data not shown), increased the baseline levels of oxidative damage (8-OHG, lipid peroxidation, protein carbonyls and 3-nitrotyrosine), and rendered cells more susceptible to death induced by serum deprivation, H2O2 treatment, exposure to MPP+ or addition of lactacystin or staurosporine, promoting more rapid loss of viability. In general, injurious conditions raised levels of oxidative damage and 3-nitrotyrosine within cells, and this was aggravated by the mutant α-synucleins (Fig. 8). One especially neurotoxic end product of lipid peroxidation is HNE and its increased formation in PD had been described (Yoritaka et al. 1996). HNE caused a loss of viability in both cell lines, which was aggravated by overexpression of mutant α-synucleins. However, the wild-type α-synuclein transfectants were also more sensitive to HNE, although less so than the mutant α-synuclein transfectants. HNE exerts its toxicity in large part by binding to proteins (Oliver et al. 1990), and it is possible that alteration of the protein composition of cells may aggravate its toxicity. Somewhat similar data were obtained with staurosporine, a protein kinase inhibitor that is widely used to induce apoptosis, accompanied by oxidative stress (Ahlemeyer and Krieglstein 2000). The mutant α-synucleins accelerated loss of viability in cells treated with staurosporine. The wild-type α-synuclein did so as well, but to a lesser degree.
We also examined the effect of lactacystin, a proteasome inhibitor. Proteasomes degrade many cellular proteins, both normal and abnormal, after ubiquitination (Sitte et al. 1998) and there is evidence for impairments of proteasome function in the neurodegenerative diseases (Halliwell and Jenner 1998). Bennett et al. (1999) reported that both wild-type and the A53T mutant α-synuclein are degraded by the proteasome in SH-SY5Y cells, but the mutant is degraded more slowly. Lactacystin addition caused apoptosis, accompanied by increased oxidative damage. A rise in protein carbonyls in lactacystin-treated cells has already been reported (Sitte et al. 1998), consistent with the degradation of oxidatively modified proteins by this mechanism. The mutant α-synucleins again accelerated lactacystin-induced cell death. To a lesser extent, so did wild-type α-synuclein. It may be that overexpression of these proteins, especially the mutants that may be degraded more slowly, cause choking of the proteasome system and disrupt cellular function. However, cells overexpressing β-galactosidase were not more sensitive to lactacystin. It may be, of course, that this protein, which is not normally expressed in NT-2 and SK-N-MC cell lines, is not degraded by the proteasome system. Interestingly, it has been suggested that HNE-modified proteins can also block the proteasome system (Grune et al. 1995), which would be consistent with the results we obtained on addition of this substance.
Our data show that over-expression of wild-type α-synuclein does not in general raise basal levels of oxidative damage or 3-nitrotyrosine in the two cell lines examined. Indeed, it was protective against cell death induced by serum withdrawal and H2O2 treatment, although not against MPP+. However, it accelerated loss of viability caused by HNE, staurosporine or lactacystin (the latter two of which are, of course, non-physiological insults). In all cases, the cell death occurred by apoptosis as evidenced by morphological examination, DNA laddering and other features of apoptosis. It seems that function of wild-type α-synuclein is cell line- and insult-specific, since many previous studies suggest both protective and non-protective properties (Da Costa et al. 2000; Masliah et al. 2000; Zhou et al. 2000).
By contrast, either of the mutant α-synucleins examined accelerated cell death induced by any of the insults examined. They also raised baseline levels of some parameters of oxidative stress. α-Synuclein is one of the abundant proteins in cells (Maroteaux et al. 1988). Data suggest that its expression in transfected cells was 3.5-fold higher than that of untransfected cells. It may result in accumulation and aggregation of normal and abnormal proteins in cytoplasm, as shown in previous studies (El-Agnaf and Irvine 2000; Mattila et al. 2000). These can impair the protein clearing system, and induce oxidative stress, reflected by decreased levels of GSH and increased levels of 8-OHG, protein carbonyls, lipid peroxidation and 3-nitrotyrosine.
Our data argue strongly that the presence of mutant α-synucleins in PD may cause enhanced sensitivity to any type of insult, leading to apoptosis associated with increased oxidative damage. Of course, whether oxidative damage is a consequence of apoptosis or a cause of it remains to be elucidated, and may differ with different insults. For example, cytochrome c released from mitochondria as a result of apoptosis may interact with α-synucleins (Hashimoto et al. 1999b). Consistent with our view, immortalized dopaminergic neurons expressing mutated α-synuclein are hypersensitive to damage by 6-hydroxydopamine (Kholodilov et al. 1999) and other insults (Saha et al. 2000), and this has been observed in studies with primary culture and transgenic mice (Da Costa et al. 2000; Ostrerova-Golts et al. 2000; Van der Putten et al. 2000; Zhou et al. 2000).