Abstract
We established previously that alpha-synuclein displayed a protective anti-apoptotic phenotype in neurons, mainly by down-regulating p53-dependent caspase-3 activation (Alves da Costa, C., Ancolio, K., and Checler, F. (2000) J. Biol. Chem. 275, 24065-24069; Alves da Costa, C., Paitel, E., Vincent, B., and Checler, F. (2002) J. Biol. Chem. 277, 50980-50984). This function was abolished by Parkinson disease-linked pathogenic mutations and by the dopaminergic toxin, 6-hydroxydopamine (6OH-DOPA) (Alves da Costa, C., Paitel, E., Vincent, B., and Checler, F. (2002) J. Biol. Chem. 277, 50980-50984). However, the mechanisms by which 6OH-DOPA interfered with alpha-synuclein function remained unclear. Here we showed that 6OH-DOPA prevents alpha-synuclein-mediated anti-apoptotic function by altering its degradation. Thus, 6OH-DOPA treatment of TSM1 neurons and SH-SY5Y neuroblastoma cells enhances endogenous alpha-synuclein-like immunoreactivity and inhibits the catabolism of endogenous and recombinant alpha-synucleins by purified 20 S proteasome. Furthermore, we demonstrated that 6OH-DOPA directly inhibits endogenous proteasomal activity in TSM1 and SH-SY5Y cells and also blocks purified proteasome activity in vitro. This inhibitory effect can be prevented by the anti-oxidant phenyl-N-butylnitrone. We also established that 6OH-DOPA triggers the aggregation of recombinant alpha-synuclein in vitro. Therefore, we conclude that 6OH-DOPA abolishes alpha-synuclein anti-apoptotic phenotype by inhibiting its proteasomal degradation, thereby increasing its intracellular concentration and potential propensity to aggregation, the latter phenomenon being directly exacerbated by 6OH-DOPA itself. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), another toxin inducer of Parkinson disease-like pathology, does not affect alpha-synuclein protective function and fails to trigger aggregation of recombinant alpha-synuclein. Furthermore, MPP(+) does not alter cellular proteasomal activity, and only high concentrations of the toxin affect purified 20 S proteasome by a mechanism that remains insensitive to phenyl-N-butylnitrone. The drastically distinct effects of 6OH-DOPA and MPP(+) on alpha-synuclein function are discussed with respect to Parkinson disease pathology and animal models mimicking this pathology.
Highlights
Parkinson disease cases are generally of sporadic origin, but a few cases have a genetic etiology
In this study we demonstrate that 6OH-DOPA, but not 1-methyl-4-phenylpyridinium (MPPϩ), interferes with the ␣-syn anti-apoptotic phenotype by both inhibiting its proteasomal degradation and promoting its aggregation
A, mock-transfected or ␣-syn-expressing TSM1 neurons were treated for 2 h with staurosporine (STS, 1 M) or for 8 h with 6OH-DOPA (6OH, 0.2 mM) or MPPϩ (0.2 mM), and active caspase-3-like immunoreactivity (Casp-3act) and tubulin expression were analyzed as described under “Experimental Procedures.”
Summary
Materials and Cells—Z-Gly-Gly-Leu-7-aminomethylcoumarin, staurosporine, lactacystin, stabilized 6OH-DOPA, phenyl-N-butylnitrone (PBN), and MPPϩ were all purchased from Sigma. Endogenous Proteasomal Activity—TSM1 cells were cultivated in 6-well plates until 70 – 80% of confluence and treated for 8 h at 37 °C with various concentrations of 6OH-DOPA (phosphate-buffered saline) or MPPϩ (dissolved in water). Degradation of Endogenous and Recombinant ␣-Syn by Purified 20 S Proteasome—TSM1 neurons overexpressing ␣-syn were cultivated in 6-well plates until confluent, harvested, centrifuged, and lysed with 10 mM Tris-HCl, pH 7.5. Fifty g of the cell homogenates were incubated for 3 or 6 h with 15 l of 20 S purified proteasome activity in the absence or in the presence of 0.2 mM 6OH-DOPA. XTT and Caspase Assays—TSM1 neurons were grown in a 5% CO2 atmosphere in 96-well plates in a final volume of 100 l per well and were incubated overnight at 37 °C in the presence and absence of various concentrations of MPPϩ. Statistical Analysis—Statistical analysis was performed with PRISM software (Graphpad Software, San Diego), by using the Newman-Keuls multiple comparison tests for one-way analysis of variance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
