Dependence of alpha-synuclein aggregate morphology on solution conditions.
Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.
Hoyer
Wolfgang
W
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Antony
Thomas
T
Cherny
Dmitry
D
Heim
Gudrun
G
Jovin
Thomas M
TM
Subramaniam
Vinod
V
D052160:Benzothiazoles; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012964:Sodium / Q000494:pharmacology; D012996:Solutions / Q000737:chemistry; D051843:Synucleins; D013844:Thiazoles / Q000378:metabolism; D051844:alpha-Synuclein
D052160:Benzothiazoles; D007477:Ions; D009419:Nerve Tissue Proteins; C497604:SNCA protein, human; D012996:Solutions; D051843:Synucleins; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T; D012964:Sodium; D008274:Magnesium