Oxidative stress-induced phosphorylation, degradation and aggregation of alpha-synuclein are linked to upregulated CK2 and cathepsin D.
Intracellular accumulation of alpha-synuclein (alpha-Syn) as filamentous aggregates is a pathological feature shared by Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, referred to as synucleinopathies. To understand the mechanisms underlying alpha-Syn aggregation, we established a tetracycline-off inducible transfectant (3D5) of neuronal lineage overexpressing human wild-type alpha-Syn. Alpha-Syn aggregation was initiated by exposure of 3D5 cells to FeCl2. The exposure led to formation of alpha-Syn inclusions and oligomers of 34, 54, 68 kDa and higher molecular weights. The oligomers displayed immunoreactivity with antibodies to the amino-, but not to the carboxyl (C)-, terminus of alpha-Syn, indicating that C-terminally truncated alpha-Syn is a major component of oligomers. FeCl2 exposure also promoted accumulation of S129 phosphorylated monomeric alpha-Syn (P alpha-Syn) and casein kinase 2 (CK2); however, G-protein-coupled receptor kinase 2 was reduced. Treatment of FeCl2-exposed cells with CK2 inhibitors (DRB or TBB) led to decreased formation of alpha-Syn inclusions, oligomers and P alpha-Syn. FeCl2 exposure also enhanced the activity/level of cathepsin D. Treatment of the FeCl2-exposed cells with pepstatin A or NH4Cl led to reduced formation of oligomers/inclusions as well as of approximately 10 and 12 kDa truncated alpha-Syn. Our results indicate that alpha-Syn phosphorylation caused by FeCl2 is due to CK2 upregulation, and that lysosomal proteases may have a role in producing truncated alpha-Syn for oligomer assembly.
Takahashi
Makio
M
Department of Neuroscience, Mayo Clinic College of Medicine, 4500 San Pablo Road, Jacksonville, FL 32224, USA.
Ko
Li-wen
LW
Kulathingal
Jayanarayan
J
Jiang
Peizhou
P
Sevlever
Daniel
D
Yen
Shu-Hui C
SH
D015153:Blotting, Western; D047390:Casein Kinase II / Q000096:biosynthesis* / Q000502:physiology*; D002402:Cathepsin D / Q000096:biosynthesis* / Q000502:physiology*; D002478:Cells, Cultured; D005296:Ferrous Compounds / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000473:pathology / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases / Q000502:physiology
D005296:Ferrous Compounds; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051844:alpha-Synuclein; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D002402:Cathepsin D; D013752:Tetracycline; C029451:ferrous chloride