Doppel-induced apoptosis and counteraction by cellular prion protein in neuroblastoma and astrocytes.
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.
Qin
K
K
Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, MSTF 700A, Baltimore, MD 21201, USA.
Zhao
L
L
Tang
Y
Y
Bhatta
S
S
Simard
J M
JM
Zhao
R Y
RY
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001253:Astrocytes / Q000187:drug effects* / Q000648:ultrastructure; D015153:Blotting, Western / Q000379:methods; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique / Q000379:methods; D058851:GPI-Linked Proteins; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D020287:In Situ Nick-End Labeling / Q000379:methods; D007211:Indoles; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D011328:Prions / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D034622:RNA Interference / Q000502:physiology; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors; D014162:Transfection / Q000379:methods
D058851:GPI-Linked Proteins; D007211:Indoles; D011328:Prions; C122036:Prnd protein, mouse; C007293:DAPI; D020169:Caspases