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Characterization of antibodies that selectively detect α-synuclein in pathological inclusions

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Abstract

Sensitive detection of α-synuclein (α-syn) pathology is important in the diagnosis of disorders like Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy and in providing better insights into the etiology of these diseases. Several monoclonal antibodies that selectively react with aggregated α-syn in pathological inclusions and reveal extensive and underappreciated α-syn pathology in the brains of diseased patients were previously reported by Duda et al. (Ann Neurol 52:205–210, 2002). We sought to characterize the specificity of some of these antibodies (Syn 505, Syn 506 and Syn 514); using C-terminal and N-terminal truncations of α-syn, all three antibodies were determined to require N-terminal epitopes that minimally comprise amino acids 2–4, but possibly extend to amino acid 12 of α-syn. The selectivity of these antibodies was further assessed using biochemical analysis of human brains and reactivity to altered recombinant α-syn proteins with duplication variants of amino acids 1–12. In addition, by expressing wild-type or a double mutant (E46K/A53T) of α-syn in cultured cells and by comparing their immunoreactivities to another antibody (SNL-4), which has a similar primary epitope, it was determined that Syn 505, Syn 506 and Syn 514 recognize conformational variants of α-syn that is enhanced by the presence of the double mutations. These studies indicate that antibodies Syn 505, Syn 506 and Syn 514 preferentially recognize N-terminal epitopes in complex conformations, consistent with the dramatic conformational change associated with the polymerization of α-synuclein into amyloid fibrils that form pathological inclusions.

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Acknowledgments

We thank Drs. John Q. Trojanowski and Virginia M.-Y. Lee (Center for Neurodegenerative Disease Research, University of Pennsylvania) and the families of patients who made this research possible.

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Authors and Affiliations

  1. Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, 123 John Morgan Building, Philadelphia, PA, 19104, USA

    Elisa A. Waxman & Benoit I. Giasson

  2. Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104, USA

    John E. Duda

  3. Parkinson’s Disease Research, Education and Clinical Center, Philadelphia Veterans Affairs Medical Center, Philadelphia, PA, 19104, USA

    John E. Duda

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  1. Elisa A. Waxman
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  2. John E. Duda
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  3. Benoit I. Giasson
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Correspondence to Benoit I. Giasson.

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This work was funded by grants from the National Institute on Aging (AG09215) and the National Institute of Neurological Disorders and Stroke (NS053488). E.A.W. was supported by a training grant (T32 AG00255) from the National Institute on Aging.

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Waxman, E.A., Duda, J.E. & Giasson, B.I. Characterization of antibodies that selectively detect α-synuclein in pathological inclusions. Acta Neuropathol 116, 37–46 (2008). https://doi.org/10.1007/s00401-008-0375-1

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  • DOI: https://doi.org/10.1007/s00401-008-0375-1

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