An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology

Institute of Neurology, Medical University of Vienna, AKH 4 J, Währinger Gürtel 18-20, 1097, Vienna, Austria.
Acta Neuropathologica (Impact Factor: 10.76). 02/2012; 124(1):37-50. DOI: 10.1007/s00401-012-0964-x
Source: PubMed


α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.

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Available from: Maria Pikkarainen
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    • "IHC methodology is prone to considerable variability that might cause differing results, as was elegantly reported by Mackenzie et al. (2006b). Today, it is acknowledged that use of the IHC methods requires knowledge and competence both regarding methodology and interpretation of the result obtained (McNicol and Richmond 1998; Shi et al. 2001; Gelpi et al. 2007; D'Amico et al. 2009; Pikkarainen et al. 2010a; Karlsson and Karlsson 2011; Kovacs et al. 2012). In addition, the agreement rate for the designation of a specific type of a disease by numerous investigators might be less optimal, as previously reported by BrainNet Europe (BNE) (Alafuzoff et al. 2008b, 2009a, b). "
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    ABSTRACT: The BrainNet Europe consortium assessed the reproducibility in the assignment of the type of frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein (TDP) 43 following current recommendations. The agreement rates were influenced by the immunohistochemical (IHC) method and by the classification strategy followed. p62-IHC staining yielded good uniform quality of stains, but the most reliable results were obtained implementing specific Abs directed against the hallmark protein TDP43. Both assessment of the type and the extent of lesions were influenced by the Abs and by the quality of stain. Assessment of the extent of the lesions yielded poor results repeatedly; thus, the extent of pathology should not be used in diagnostic consensus criteria. Whilst 31 neuropathologists typed 30 FTLD-TDP cases, inter-rater agreement ranged from 19 to 100 per cent, being highest when applying phosphorylated TDP43/IHC. The agreement was highest when designating Type C or Type A/B. In contrast, there was a poor agreement when attempting to separate Type A or Type B FTLD-TDP. In conclusion, we can expect that neuropathologist, independent of his/her familiarity with FTLD-TDP pathology, can identify a TDP43-positive FTLD case. The goal should be to state a Type (A, B, C, D) or a mixture of Types (A/B, A/C or B/C). Neuropathologists, other clinicians and researchers should be aware of the pitfalls whilst doing so. Agreement can be reached in an inter-laboratory setting regarding Type C cases with thick and long neurites, whereas the differentiation between Types A and B may be more troublesome.
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    • "We recently reported that this antibody has strong selectivity for β-sheet rich α-synuclein oligomers and that the ependymal α-synuclein immunoreactivity correlates well with Braak stages of Lewy-related pathology [1]. The antibody is suitable for detecting α-synuclein pathology even in tissue fixed for a very long time in formalin [2]. We report here that 5G4 antibody is able to detect disease-associated α-synuclein in the cerebrospinal fluid of individuals with neuropathologically proven α-synuclein deposition in the brain [3]. "

    Full-text · Article · Sep 2014 · Clinical neuropathology
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    • "We recently described an antibody against α-synuclein (named 5G4), which shows high specificity for the disease-associated forms, including high molecular weight fraction of β-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed [15, 16]. Furthermore, we have demonstrated that d-α-syn deposits in the ependymal layer of the ventricles and aqueduct [15] in PD and DLB, thus it is likely that it can be detected in the CSF. "
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    ABSTRACT: With the aim to evaluate the significance and reliability of detecting disease-specific α-synuclein in the cerebrospinal fluid (CSF) we developed an ELISA and bead-assay. We used a commercial antibody (5G4) that does not bind to the physiological monomeric form of α-synuclein, but is highly specific for the disease-associated forms, including high molecular weight fraction of β-sheet rich oligomers. We applied both tests in CSF from a series of neuropathologically confirmed αsynucleinopathy cases, including Parkinson' disease dementia (PDD) and dementia with Lewy bodies (DLB) (n = 7), as well as Alzheimer' disease (n = 6), and control patients without neurodegenerative pathologies (n = 9). Disease-specific α-synuclein was detectable in the CSF in a subset of patients with α-synuclein pathology in the brain. When combined with the analysis of total α-synuclein, the bead-assay for disease-specific α-synuclein was highly specific for PDD/DLB. Detection of disease-associated αsynuclein combined with the total levels of α-synuclein is a promising tool for the in-vivo diagnosis of αsynucleinopathies, including PDD and LBD.
    Full-text · Article · Sep 2014 · Clinical neuropathology
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