Identifiying the Anti-Tr Antigen in Paraneoplastic Cerebellar Degeneration (IN3-1.003)

Abstract

Objective: We aimed to identify the long sought antigen recognized by anti-Tr antibodies. Background Paraneoplastic cerebellar degeneration (PCD) is characterized by subacute cerebellar ataxia which coincides with the presence of specific tumour types and antineuronal antibodies. One particular form of PCD is associated with Hodgkin Lymphoma (HL) and the presence of antibodies against cerebellar Purkinje cells in several of these patients9 sera. These later called anti-Tr antibodies recognize a specific punctuate immunoreactivity in the large dendritic tree as well as the soma of the Purkinje cells, but not in the axons. Although this characteristic immunoreactive pattern is considered to be a good hallmark for the presence of anti-Tr antibodies, further diagnosis is elaborate. In order to aid this diagnosis, and to understand the pathogenic nature of the PCD we aimed to identify the antigen recognized by anti-Tr antibodies. Design/Methods: To identify the antigen we performed immunoprecipitation using four anti-Tr positive sera on total rat brain extract followed by mass spectrometry. By Western blotting and cell-based assays we subsequently determined the region of the epitope recognized by the anti-Tr antibodies. Deletion and mutant constructs were generated to further map the antigenic region. Results: We identified Delta/Notch-like epidermal growth factor (EGF)-related Receptor (DNER) as the Tr antigen. In a cell-based screening assay 191 control samples were negative, whereas all of the 12 anti-Tr positive sera stained HA-tagged-DNER expressing cells. Using deletion constructs we pinpointed the main epitope to the extracellular domain. Glycosylation inhibitor tunicamycin and N-glycosylation mutations in DNER abolished the anti-Tr staining, indicating that DNER must be glycosylated to be recognized by anti-Tr antibodies. Conclusions: Anti-Tr positive sera recognize DNER. Using the cell based assay, presence of anti-Tr antibodies in patients with PCD and HL can now be screened both quickly and reliably. Supported by: The Netherlands Organization for Scientific Research (NWO-ALW-VICI, C.H.), the Netherlands Organization for Health Research and Development (ZonMW-TOP, C.H.), the European Science Foundation (EURYI, C.H.). Disclosure: Dr. Sillevis Smitt has nothing to disclose. Dr. van den Berg has nothing to disclose. Dr. Maat has nothing to disclose. Dr. Hulsenboom has nothing to disclose. Dr. van den Berg has nothing to disclose. Dr. Demmers has nothing to disclose. Dr. Hoogenraad has nothing to disclose.