Pathobiology of VPS45 severe congenital neutropenia

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Mutations in the endosomal-lysosomal regulatory protein VPS45 cause a life-threatening form of severe congenital neutropenia (SCN) characterized by agranulocytosis, neutrophil dysfunction, platelet alpha granule deficiency, and myelofibrosis. Normal function of neutrophils and platelets requires formation, mobilization and fusion of exocytic granules; when dysregulated, these defects lead to an “intracellular traffic jam” in VPS45 neutropenia and other granule disorders. VPS45 regulates the transport and assembly of endosomal- lysosomal SNARE complexes for SNARE-mediated membrane fusion. We hypothesize that mutant VPS45 protein misregulates SNARE complexes in mammalian hematopoietic cells, leading to neutropenia and neutrophil dysfunction through abnormal intracellular protein trafficking and granule function. To test this hypothesis, we have developed in vitro and in vivo systems for biochemical and physiological analysis of SCN caused by VPS45 mutations, including development and initial characterization of the first mouse model for neutropenia due to an endosomal-lysosomal defect. Our Specific Aims are: 1. Investigate the biochemical impact of Vps45 mutations on SNARE binding in vitro and organelle trafficking in yeast. We are quantitatively assessing binding of recombinant wild-type (wt) and mutant yeast Vps45 and human VPS45 proteins to cognate SNARE proteins and other partners, and measuring the rates of SNARE complex assembly and membrane fusion. We are measuring effects in yeast on Vps45 and SNARE protein levels, endo- and exocytosis, trafficking of endosomal and vacuolar proteins, recruitment of Vps45 to membranes, and binding of Vps45 to partner proteins. 2. Determine the molecular consequences of VPS45 mutations in neutrophils and fibroblasts in vitro. We are testing the “intracellular traffic jam” hypothesis in mammalian cells by examining the effects of pathogenic VPS45 mutations on SNARE assembly, membrane trafficking, and granule biogenesis and fusion. 3. Determine the functional consequences of VPS45 dysfunction in mouse and patient cells. We are investigating whether abnormalities in membrane trafficking and granule biogenesis lead to defects in the function of mouse and human cells.. We will focus on pathways leading to apoptosis and to defects in phagocytic function. 4. Test the physiological consequences of VPS45 dysfunction in our mouse model of VPS45 neutropenia. We are investigating the effects of VPS45 pathogenic mutations on neutrophil number, bone marrow architecture, organ pathology, and resistance to infection, in order to help determine how VPS45 dysfunction affects neutrophil function and host defense in the whole organism. The proposed collaborative studies, combining the complementary expertise of Drs. Munson and Newburger, will elucidate previously unexplored molecular mechanisms for the novel SCN caused by VPS45 mutations, while gaining insights into this and other syndromes caused by vesicle and granule defects.