Research reportImmunoelectron-microscopic demonstration of NACP/α-synuclein-epitopes on the filamentous component of Lewy bodies in Parkinson's disease and in dementia with Lewy bodies
Introduction
NACP, the precursor protein of non-Aβ component (NAC) of Alzheimer's disease amyloid [17], was demonstrated to be a presynaptic protein [9], and was later named human α-synuclein independently [10]. Recently, a G209A mutation was identified in the NACP gene in chromosome 4, from four families with Parkinson's disease (PD) [13], although subsequent reports failed to substantiate this and claimed that the NACP mutation was not a chief cause for the majority of the familial PD [19]. More recently, however, another missense G88C mutation was identified in the NACP gene in an independent familial PD pedigree [11]. As had been expected, Lewy bodies (LBs) and Lewy neurites were found to show immunoreactivity to antibodies against NACP protein in PD and in dementia with Lewy bodies (DLB) 15, 18, suggesting NACP to be a candidate protein primarily involved in the formation of these abnormal structures. Lewy bodies and pale bodies (PBs) are two interrelated neuronal inclusions of PD 2, 3, 4. In this study, we examined brains of patients with PD and DLB by using antibodies raised against NACP, and we found that filamentous components (LB-filaments) were immunolabeled in LBs, PBs, and thread-like structures in neuronal perikarya. Further detailed electron-microscopic (EM) observation should add new insights to our knowledge of LB formation.
Section snippets
Tissue source and section preparation
Six brain samples from Japanese patients with PD, DLB, and elderly patients with occasional Lewy bodies were obtained from the Department of Laboratory Medicine, National Center Hospital for Mental, Nervous, and Muscular Disorders, NCNP. Three patients had Parkinson's disease (a 58-year old female, a 78-year-old male, and a 79-year-old female; autopsy Nos. 706, 635, and 721, respectively), one patient had DLB (a 92-year-old male; autopsy No. 572), and two patients without neurodegenerative
Characterization and specificity of primary antibodies
Immunoblot analyses revealed that all of the anti-NACP antibodies (MDV2, EQV1, PQE3) recognized the recombinant NACP, but the anti-β-synuclein antibody (REE1) did not react with NACP. Furthermore, the immunoreactivity was abolished when incubated with each of the pre-absorbed antibody solutions (Fig. 1A).
Immunoblotting of the cytosolic fraction of rat cortex showed that EQV1 detected NACP, having a molecular mass of ca. 19 kDa (Fig. 1B, lane 1) [17], while the anti-β-synuclein-specific antibody
Characterization of anti-NACP antibodies
In this study, we have employed three anti-NACP antibodies, named MDV2, EQV1, and PQE3, that were raised against peptides corresponding to partial sequences of the N-terminal region, NAC domain, and C-terminal region of NACP, respectively.
Our immunoblot analyses, using recombinant NACP and a rat brain cytosolic fraction, demonstrated that MDV2 recognized both NACP and β-synuclein; EQV1 and PQE3 identified NACP specifically; and REE1 detected β-synuclein selectively. Since the amino-acid
Acknowledgements
The authors are grateful to Prof. Masanori Kurokawa, Prof. Yasuo Ihara, and Dr. Kazuhiko Ikeda for their critical comments on the manuscript, and to Mr. Y. Shoda, Ms. M. Kato, Mr. O. Nakajima, and Ms. A. Sakamoto for their excellent technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research (C:08671119 to K.A. and C:09680780 to K.U.) from the Ministry of Education, Science, and Culture, Japan.
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