Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility

Roberto De Giorgio, Monica Bovara, Giovanni Barbara, Marco Canossa, Giovanni Sarnelli, Fabrizio De Ponti, Vincenzo Stanghellini, Marcello Tonini, Silvia Cappello, Eleonora Pagnotta, Eduardo Nobile-Orazio, Roberto Corinaldesi

Research output: Contribution to journalArticle

Abstract

Background & Aims: The role of autoimmunity underlying paraneoplastic gut dysmotility remains unsettled. Because anti-Hu antibodies may impair enteric neuronal function, we tested whether anti-HuD-positive sera from patients with paraneoplastic gut dysmotility or commercial anti-HuD antibodies activated the apoptotic cascade in a neuroblastoma cell line and cultured myenteric neurons. Methods: Anti-HuD antibodies from patients with severe paraneoplastic gut dysmotility were characterized by immunofluorescence and immunoblot. SH-Sy5Y neuroblasts and cultured myenteric neurons were exposed to sera containing anti-HuD antibodies or 2 commercial anti-HuD antibodies. Cells were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique to evaluate apoptosis. Immunofluorescence was used to identify activated caspase-3 and apaf-1, along with microtubule-associated protein 2. Results: In SH-Sy5Y cells, the percentage of TUNEL-positive nuclei observed after exposure to anti-HuD-positive sera (32% ± 7%) or anti-HuD antibodies (23% ± 2%) was significantly greater than that of control sera or fetal calf serum (P <0.001). The time-course analysis showed a significantly greater number of apoptotic neuroblastoma cells evoked by the 2 commercial anti-HuD antibodies at 24, 48, and 72 hours versus controls. The number of TUNEL-positive myenteric neurons exposed to anti-HuD antibodies (60% ± 14%) was significantly greater than that of fetal calf serum (7% ± 2%; P <0.001). Apaf-1 and caspase-3 immunolabeling showed intense cytoplasmic staining in a significantly greater proportion of cells exposed to anti-HuD-positive sera or to commercial anti-HuD antibodies compared with controls. Conclusions: Anti-HuD antibodies evoked neuronal apoptosis that may contribute to enteric nervous system impairment underlying paraneoplastic gut dysmotility. Apaf-1 activation suggests participation of a mitochondria-dependent apoptotic pathway.

Original languageEnglish
Pages (from-to)70-79
Number of pages10
JournalGastroenterology
Volume125
Issue number1
DOIs
Publication statusPublished - Jul 1 2003

ASJC Scopus subject areas

  • Gastroenterology

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    De Giorgio, R., Bovara, M., Barbara, G., Canossa, M., Sarnelli, G., De Ponti, F., Stanghellini, V., Tonini, M., Cappello, S., Pagnotta, E., Nobile-Orazio, E., & Corinaldesi, R. (2003). Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility. Gastroenterology, 125(1), 70-79. https://doi.org/10.1016/S0016-5085(03)00664-4