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ABSTRACT: Proteasome inhibition and aggregation in Parkinson's disease: a comparative study in untransfected and transfected cells Emiliano Biasini*,,1, Luana Fioriti*,,1, Ilaria Ceglia*, Roberto Invernizzi*, Alessandro Bertoli*,2, Roberto Chiesa*, and Gianluigi Forloni* * Department of Neuroscience, Istituto di Ricerche Farmacologiche ‘Mario Negri’ Milano, Italy Dulbecco Telethon Institute (DTI), Istituto di Ricerche Farmacologiche ‘Mario Negri’ Milano, Italy Address correspondence and reprint requests to Gianluigi Forloni Istituto di Ricerche Farmacologiche ‘Mario Negri’, 20157 Milano, Italy. E-mail: [log in to unmask] Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in Parkinson's disease (PD) and other neurodegenerative disorders. We have investigated the effect of UPS inhibition on the metabolism of -synuclein (SYN) and parkin, two proteins genetically and histopathologically associated to PD. Pharmacological inhibition of proteasome induced accumulation of both parkin and SYN in transfected PC12 cells. We found that this effect was caused by increased protein synthesis rather than impairment of protein degradation, suggesting that inhibition of the UPS might lead to non-specific up-regulation of cytomegalovirus (CMV)-driven transcription. To investigate whether endogenous parkin and SYN can be substrate of the UPS, untransfected PC12 cells and primary mesencephalic neurones were exposed to proteasome inhibitors, and parkin and SYN expression was evaluated at both protein and mRNA level. Under these conditions, we found that proteasome inhibitors did not affect the level of endogenous parkin and SYN. However, we confirmed that dopaminergic neurones were selectively vulnerable to the toxicity of proteasome inhibitors. Our results indicate that studies involving the use of proteasome inhibitors, particularly those in which proteins are expressed from a heterologous promoter, are subjected to potential artefacts that need to be considered for the interpretation of the role of UPS in PD pathogenesis. Key Words: mesencephalic cultures – parkin – Parkinson's disease – protein misfolding – synuclein – ubiquitin- proteasome Abbreviations used: Act D, actinomycin D; ALLN, N-acetyl-Leu-Leu-Norleu-al; CHX, cycloheximide; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; LB, Lewy bodies; MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium; PC12, pheocromocytoma cells; PD, Parkinson's disease; SYN, -synuclein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; UPS, ubiquitin-proteasome system SOURCE: Journal of Neurochemistry, Vol. 88, No. 3, 2004 545-553 http://www.jneurochem.org/cgi/content/abstract/88/3/545 * * * ---------------------------------------------------------------------- To sign-off Parkinsn send a message to: mailto:[log in to unmask] In the body of the message put: signoff parkinsn