Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons.
Alpha-synuclein accumulates in the brains of sporadic Parkinson's disease patients as a major component of Lewy bodies, and mutations in alpha-synuclein are associated with familial forms of Parkinson's disease. The pathogenic mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson's disease-associated mutations alter the subcellular distribution of alpha-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP fusion proteins are partially truncated to a approximately 27-kDa form. This non-fluorescent carboxy-terminally modified fusion protein spontaneously forms inclusions in the neuronal cytoplasm. A marked increase in the accumulation of inclusions is detected following treatment with each of three proteasome inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the idea that the Ala30Pro alpha-synuclein protein conformation differs from wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By contrast, overexpression of unmodified alpha-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our finding that manipulations of the carboxy-terminus of alpha-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of alpha-synuclein aggregation in Lewy bodies.
McLean
P J
PJ
Alzheimer's Disease Research Unit, Department of Neurology, Rm 6405, Massachusetts General Hospital East, 149, 13th Street, Charlestown, MA 02129, USA.
Kawamata
H
H
Hyman
B T
BT
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D002352:Carrier Proteins / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003451:Cryoprotective Agents / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005333:Fetus; D017930:Genes, Reporter / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000378:metabolism* / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D051843:Synucleins; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051844:alpha-Synuclein
D002352:Carrier Proteins; D003451:Cryoprotective Agents; D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C497604:SNCA protein, human; C118958:SNCAIP protein, human; C497605:Snca protein, mouse; C498464:Sncaip protein, mouse; D051843:Synucleins; D051844:alpha-Synuclein; C063261:acetylleucyl-leucyl-norleucinal; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D004121:Dimethyl Sulfoxide