Abstract
Lewy bodies, neuropathological hallmarks of Parkinson's disease and dementia with Lewy bodies, comprise alpha-synuclein filaments and other less defined proteins. Characterization of Lewy body proteins that interact with alpha-synuclein may provide insight into the mechanism of Lewy body formation. Double immunofluorescence labeling and confocal microscopy revealed approximately 80% of cortical Lewy bodies contained microtubule-associated protein 1B (MAP-1B) that overlapped with alpha-synuclein. Lewy bodies were isolated using an immunomagnetic technique from brain tissue of patients dying with dementia with Lewy bodies. Lewy body proteins were resolved by polyacrylamide gel electrophoresis. Immunoblotting confirmed the presence of MAP-1B and alpha-synuclein in purified Lewy bodies. Direct binding studies revealed a high affinity interaction (IC(50) approximately 20 nm) between MAP-1B and alpha-synuclein. The MAP-1B-binding sites were mapped to the last 45 amino acids of the alpha-synuclein C terminus. MAP-1B also bound in vitro assembled alpha-synuclein fibrils. Thus, MAP-1B may be involved in the pathogenesis of Lewy bodies via its interaction with monomeric and fibrillar alpha-synuclein.
Highlights
Lewy bodies, neuropathological hallmarks of Parkinson’s disease and dementia with Lewy bodies, comprise ␣-synuclein filaments and other less defined proteins
A quantitative analysis conducted in sections from three case of dementia with Lewy body indicated that more than 80% of cortical Lewy bodies and neurites contained microtubule-associated protein 1B (MAP-1B) immunoreactivity
Numerous proteins have been localized in Lewy bodies mostly by immunohistochemistry [28], and one could argue that MAP-1B is just one among many of these proteins
Summary
Human full-length ␣-synuclein was expressed in Escherichia coli and purified as described previously [11], except that anion exchange chromatography was performed on a Poros HQ resin at a flow rate of 4 ml/min (Perseptive Biosystems, Allerød, Denmark). Sheep IgG was raised against a synthetic peptide corresponding to a C-terminal portion of human ␣-synuclein (residues 116 –131) and affinity purified using the peptide bound to thiopropyl-Sepharose 6B (Amersham Pharmacia Biotech). The specificity for ␣-synuclein was confirmed by absorption of the antibody using the peptide or recombinant human ␣-synuclein (data not shown). Antibody AB97/8 was raised against a synthetic peptide corresponding to the C-terminal amino acid residues 116 –131 of human ␣-synuclein. Antibody ASY-3 was raised against a synthetic peptide corresponding to amino acid residues 1–31 of ␣-synuclein. The latter two antibodies were affinity purified using immobilized recombinant ␣-synuclein. Fluorescein isothiocyanatedonkey anti-sheep, Cy3-donkey anti-mouse, biotin-conjugated donkey anti-sheep IgG, and Cy3-conjugated streptavidin were from Jackson ImmunoResearch Laboratories; horseradish peroxidase-conjugated swine anti-rabbit IgG was from Dakopatt (Glostrup, Denmark), and goat anti-rabbit IgG conjugated to 15 nm diameter gold particles was from Amersham Pharmacia Biotech
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