Molecular Mechanisms Associated with Canine Cyclic Hematopoiesis

Abstract

Canine cyclic hematopoiesis (CH) is an autosomal recessive disorder characterized by 12-14 day neutrophil cycles, a diluted coat color, and platelet storage pool disease. Canine CH is caused by a mutation in AP3B1, encoding the ß3A subunit of adaptor protein complex – 3 (AP-3). AP-3 is a protein complex responsible for intracellular protein trafficking from the trans-Golgi network (TGN)/endosome to lysosomes and secretory lysosome-like granules. Canine CH is a model of human cyclic neutropenia/severe congenital neutropenia caused by mutations in the neutrophil elastase (NE) gene ELA2. In this dissertation, it was hypothesized that NE processing/trafficking was affected by the ß3A gene mutation in CH dogs. To study canine NE processing and trafficking, both polyclonal and monoclonal antibodies against two immunogenic peptides of canine NE were generated. The generated antibodies were characterized that recognize precursor (ELA269) or mature (ELA85) canine NE. These antibodies were used in western blot and immunocytochemistry analysis in this study. Results showed that neutrophils from the CH dog accumulated large amounts of NE precursor proteins. Granule-localized mature NE enzymes were significantly reduced in the CH dog. Corresponding to the accumulation of NE precursor proteins, protein expression of BiP/GRP78, a sensor of the unfolded protein response (UPR) and a chaperone which promotes folding and prevents aggregation of nascent proteins, was increased >5 fold in neutrophils from the CH dog. Up-regulation of BiP/GRP78 was demonstrated in in vitro cultured bone marrow mononuclear cells (BMMCs) stimulated with stem cell factor and granulocyte-colony stimulating factor in the CH dog. In addition, these cultured BMMCs from the CH dog showed increased apoptosis (20%) compared to normal dogs. The induction of the UPR and apoptotic cell death of hematopoietic progenitor cells due to inefficient NE processing/trafficking represent a novel mechanism associated with neutropenia in CH dogs. Collectively, these studies further characterize the pathoetiology of canine CH and describe likely molecular mechanisms related with this disorder.