Extensive nuclear localization of alpha-synuclein in normal rat brain neurons revealed by a novel monoclonal antibody.
Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.
Yu
S
S
Key Laboratory on Neurodegenerative Diseases of Ministry of Education, Beijing Institute of Geriatrics and Xuanwu Hospital of the Capital University of Medical Sciences, Beijing 100053, China.
Li
X
X
Liu
G
G
Han
J
J
Zhang
C
C
Li
Y
Y
Xu
S
S
Liu
C
C
Gao
Y
Y
Yang
H
H
Uéda
K
K
Chan
P
P
D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity / Q000276:immunology; D001921:Brain / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D016253:Microscopy, Immunoelectron; D009474:Neurons / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D051844:alpha-Synuclein / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D051844:alpha-Synuclein