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

Abstract

Sensitive detection of alpha-synuclein (alpha-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 alpha-syn in pathological inclusions and reveal extensive and underappreciated alpha-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 alpha-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 alpha-syn. The selectivity of these antibodies was further assessed using biochemical analysis of human brains and reactivity to altered recombinant alpha-syn proteins with duplication variants of amino acids 1-12. In addition, by expressing wild-type or a double mutant (E46K/A53T) of alpha-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 alpha-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 alpha-synuclein into amyloid fibrils that form pathological inclusions.

Fig. 1

Immunohistochemistry and biochemical analysis of DLB brains with monoclonal antibodies that selectively detect pathological α-syn. a Immunostaining with Syn 505, Syn 506 and Syn 514 in the cingulate cortex of a DLB brain detecting LBs (arrows), but far greater neuritic inclusions and smaller “dot-like” aggregates (arrowheads) and relatively weak reactivity for the normal neuropil distribution of α-syn. (Bar scale = 50 μm). b Comparative immunoblotting analysis of biochemical brain samples using antibodies Syn 208 and Syn 505. The tissue from four controls (C1-4) and four DLB patients (D1-4) were fractionated biochemically as described in “Materials and methods.” HS-soluble and Triton-insoluble extracts were resolved on SDS-polyacrylamide gels and Western blots were probed with either Syn 208 or Syn 505. α-Syn was detected in the HS-fraction from all brains using either Syn 208 or Syn 505. Syn 208 detected monomeric α-syn predominantly in the Triton-insoluble extracts from DLB patients. Higher molecular mass α-syn aggregates, some that did not enter the resolving gel (arrow), were also weakly detected. Syn 505 also revealed Tritoninsoluble monomeric α-syn in diseased brains, but, in contrast, the major species detected were higher molecular mass α-syn aggregates that did not enter the resolving gel (arrow)

Fig. 2

Antibodies Syn 505, Syn 506 and Syn 514 recognize N-terminal α-syn epitopes. Western blot analysis using a recombinant carboxy-terminal truncated α-syn proteins or b GST/carboxy-truncated α-syn fusion proteins were performed as described in “Material and methods.” c Peptide mapping using ELISA analysis and synthetic peptides corresponding to various amino acids stretches within α-syn demonstrating that the minimal required epitopes for Syn 505, Syn 506 and Syn 514 comprises residues 2-4, and may extend through amino acid 12. d Immunoblotting analysis for reactivity of Syn 505, Syn 506 and Syn 514 with human (H), murine (M) or canary (C) α-syn. e Immunoblot analysis to assess the reactivity of Syn 505, Syn 506 and Syn 514 with human α-syn, β-syn or γ-syn

Fig. 3

Immunoblotting characterization of Syn 505, Syn 506 and Syn 514 using recombinant α-syn protein with altered N-terminus. a Diagram of the N-terminal altered proteins. Protein 1-12 α-syn was generated as described in “Material and methods” so that the amino acid sequence of residues 1-12 was duplicated in tandem at the N-terminus. Protein 12-1 α-syn was generated so that residues 1-12 are present before the normal N-terminal, but in the reverse orientation. b Immuno-blotting analysis with antibodies SNL-4, Syn 505, Syn 506, Syn 514, and Syn 211. Two-times the relative concentration of WT α-syn was resolved on SDS-polyacrylamide gels to account for the possible double-epitope of the 1-12 and 12-1 α-syn protein. Antibody Syn 211, which reacts with an epitope at the C-termimus of α-syn and would not be affected by these N-terminal alterations, was used as a control. Surprisingly, the analysis revealed that Syn 505, 506, and 514 antibodies recognized WT α-syn with approximately 10-fold greater immunore-activity than 1-12 α-syn, and these antibodies could not detect 12-1 α-syn. SNL-4, a polyclonal antibody raised to amino acid sequence 2-12 in α-syn, was used for comparison. SNL-4 reacted similarly with WT α-syn and 1-12 α-syn, but very weakly with 12-1 α-syn

Fig. 4

Analyses of the polymerization of A53T and E46K/A53T α-syn mutations. Recombinant A53T and E46K/A53T α-syn were incubated at 1 mg/ml for 72 h, as described in “Materials and methods.” a Representative Coomassie blue stained SDS-polyacrylamide showing the proteins in the soluble fractions (S) or in the pellets (P) following sedimentation analysis, and quantitative summary (* P = 0.003, n =7). b K114 amyloid fluorometry analyses (** P = 0.001, n = 7) demonstrating the increased propensity of E46K/A53T α-syn to polymerize compared to A53T α-syn. Data represent averages ± S.E.M

Fig. 5

Double-immunofluorescence analysis of QBI293 cells transfected with WT or E46K/A53T α-syn. Representative fields of QBI293 cells transfected with WT α-syn (a, c, d) or E46K/A53T α-syn (b, d, f). Cells were immunostained with either Syn 505 (a, b), Syn 506 (c, d), or Syn 514 (e, f) (each in red, left) and with SNL-4 (green, center). Increased single-label immunofluorescence (left panels) and double-label immunofluorescence (merge, right panels) with Syn 505, Syn 506, and Syn 514 were noted in E46K/A53T α-syn over WT α-syn transfected cells. Representative images were chosen from a single experiment. (Bar scale = 200 μm)

Fig. 6

Summary of double-immunofluorescence analyses of QBI293 cells transfected with WT or E46K/A53T α-syn. Percent of SNL-4 labeled cells that were co-labeled with Syn 505, 506, or 514. Data represent averages ± S.E.M. Comparisons demonstrate increased double-labeling in E46K/A53T α-syn transfected cells. (* P = 0.02, n = 5; ** P < 0.0001, n =4; # P = 0.05, n =6)