|Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1995: ¥1,600,000 (Direct Cost: ¥1,600,000)
In the paraneoplastic synrome which is characterized by development of malignant tumors followed by neurological symptoms, autoantibodies raised against the central nervous system (CNS) are frequently detected. Among these autoantibodies, anti-Hu, anti-Yo and anti-Ri antibodies are well studied. It is not clear, however, whether or not these antineuronal autoantibodies cause neurological disorders in patients with the syndrome. We have found a new antineuronal autoantibody in a patient with subacute sensory neuropathy and invasive ductal mammary carcinoma. To identify antigen (s) recognized by the autoantibody we have carried out Western blotting, subcellular fractionations, immunoprecipitation, immunohistochemistry and immunoelectrone microscopy. The putative antigen revealed had a molecular mass of 130 kDa on SDS-PAGE,was localized in CNS,being concentrated in nerve terminals, and was present in cytosolic fractions of brain homogenates as well as in synaptic plasma membrane fracitons. These characteristic features lead us to a protein, amphiphysin, which was identified recently as a novel synaptic vesicle-associated protein. Amphiphysin was also described recently as an autoantigen of Stiff-Man Syndrome with breast cancer. We studied whether or not our autoantibody recognizes amphiphysin by Western blotting using amphiphysin fragments expressed as GST-fusion proteins (in collaboration with Prof.P.De Camilli). A C-terminal fragment of the protein was recognized by the autoantibody. We cloned cDNA fragments of rat amphiphysin by PCR amplification of a rat brain stem cDNA library using primers complementary to a conserved region between human and chicken amphiphysin. Amphiphysin of rat was highly homologous to the human counterpart. In the retina, several bands were recognized by the antibody on Western blot, indicating the presence of isoforms. We are currently investigating the isoforms of amphiphysin.