Induction of alpha-synuclein aggregation by intracellular nitrative insult.
Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.
Paxinou
E
E
Stokes Research Institute and Department of Biochemistry and Biophysics, Children's Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Chen
Q
Q
Weisse
M
M
Giasson
B I
BI
Norris
E H
EH
Rueter
S M
SM
Trojanowski
J Q
JQ
Lee
V M
VM
Ischiropoulos
H
H
D000643:Ammonium Chloride / Q000378:metabolism / Q000493:pharmacokinetics; D002460:Cell Line; D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism / Q000494:pharmacology; D051843:Synucleins; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism; D051844:alpha-Synuclein; D051846:beta-Synuclein
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D009566:Nitrates; D016877:Oxidants; C497604:SNCA protein, human; C497607:SNCB protein, human; D051843:Synucleins; D014452:Ubiquitins; D051844:alpha-Synuclein; D051846:beta-Synuclein; D000643:Ammonium Chloride; D013481:Superoxides; C039642:peroxynitric acid; D009569:Nitric Oxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex