Doppel-induced apoptosis and counteraction by cellular prion protein in neuroblastoma and astrocytes

Abstract

Expression of a prion-like protein, doppel, induces apoptosis-like changes in cerebellar neuronal granule and Purkinje cells of prion-knockout mice and this effect can be rescued by re-introduction of cellular prion. Since most of those studies were done in transgenic mice, in the present study, we have established a murine neuro-2a cell line and the primary rat adult reactive astrocyte model for studying doppel-induced apoptosis and possible prion counteraction. We demonstrate that expression of doppel in neuro-2a cells causes apoptosis, during which DNA fragmentation occurs as visualized by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining and other intracellular changes characteristic of apoptosis are observed in the electron microscope. Using immunoblot analyses, we further demonstrate that doppel expression activates caspase-10 as well as caspase-3, but does not activate caspase-9. Addition of purified doppel to cultures of neuro-2a cells and the primary astrocytes causes similar apoptotic changes. Significantly, apoptosis induced by doppel is enhanced when cellular prion protein is depleted by RNA interference, suggesting a protective effect of cellular prion against doppel-induced apoptosis. The antagonistic interaction between cellular prion and doppel appears to involve direct protein–protein interaction possibly on cell membrane as cellular prion and doppel physically interact with each other and co-localize on cell membranes. Together, our data show that doppel induces apoptosis in neuroblastoma neuro-2a and rat primary astrocytes via a caspase-10 mediated pathway and that this effect is counteracted by cellular prion through direct interaction with doppel possibly on cell membrane.