Extensive nuclear localization of α-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 α-synuclein (α-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 α-SYN. One antibody named 3D5 recognized amino acids 115–121 of α-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 α-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of α-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic α-SYN but only 3D5 detected the nuclear α-SYN. The nuclear localization of α-SYN was further confirmed (1) by Western blot analysis in isolated nuclear fraction where the same size of α-SYN was detected, and (2) 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 α-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.