Cyclic hematopoiesis (CH) is an unusual disorder of the hematopoietic stem cell (HSC) with on/off proliferation of HSCs that causes cycles of circulating blood cells. CH is caused by mutations in the ela 2 gene encoding neutrophil elastase in human beings and in the AP3B1 gene which encodes the B1 subunit of the adaptin AP3 in dogs. Mutations in ela 2 also cause severe congenial neutropenia, Kostmann's Syndrome. Canine CH is always associated with a diluted coat color, mild bleeding disorder due to platelet dense granule storage pool disease and cutaneous mast cell deficiency. This is consistent with the notion that AP3 is a cargo protein shuttle for lysosome like organelles such as neutrophil primary granules, platelet dense granules, melanosomes and mast cell granules. The mechanism whereby mutations in ela 2 and AP3B1 result in cyclic hematopoiesis are not well understood. We hypothesize that abnormal elastase trafficking to the plasma membrane causes increased proteolytic degradation of membrane receptors and a dampened proliferative response in myeloid progenitor cells which unmasks the inherent cyclic nature of hematopoiesis. The long range goal of this project is to determine the cellular and biochemical mechanisms causing cyclic hematopoiesis with ela 2 and AP3B1 mutations.
The specific aims are 1) characterize B3A expression and function in normal and CH dogs. 2) Define the temporal regulation and intracellular trafficking of elastase during the 14 day cycle in CH dogs. 3) Determine if gene transfer of the normal B3A cDNA into CD34+ c-kit+ lin- cells corrects the cellular defects in HSCs. Cells will be transduced ex vivo with a lentiviral vector containing the normal canine B3A cDNA. Elastase trafficking to the lysosome and plasma membrane and assembly of the AP3 tetrameric complex will be determined before and after gene transfer. We will also perform autologous bone marrow transplants (n=4) with the B3A transduced cells to determine if cyclic hematopoiesis is corrected. 4) Determine if elastase trafficking to the plasma membrane causes proteolytic degradation of the c-kit membrane receptor. Western blot, receptor binding kinetics and flow cytometry will be used to determine if there is increased proteolysis or receptor recycling in CH dogs. 5) Determine if there is a platelet dense granule deficiency or abnormal storage function by the mepacrine labeling technique, determine if AP3 interacts with rab27a, a small GTPase protein necessary for dense granule storage function and screen for additional proteins interacting with AP3 by co-immunoprecipitation and Western blotting. 6) Determine if HSCs are regulated in a cell-autonomous fashion (Chalone hypothesis) or in a paracrine manner by a diffusable factor using nonmyeloablative bone marrow transplantation to establish mixed hematopoietic chimerism with DLA-identical sex-mismatched normal and CH dogs (n=6). A Y-chromosome PCR assay will be used to determine the % normal and CH cells in circulation. These studies will advance our understanding of the basic biology of HSCs, granule formation and protein trafficking using the CH dog model uniquely available in our laboratory.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL079569-04
Application #
7275291
Study Section
Special Emphasis Panel (ZHL1-CSR-D (S1))
Program Officer
Qasba, Pankaj
Project Start
2004-09-30
Project End
2009-07-31
Budget Start
2007-08-01
Budget End
2008-07-31
Support Year
4
Fiscal Year
2007
Total Cost
$311,477
Indirect Cost
Name
University of Alabama Birmingham
Department
Biochemistry
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Vallejo, Matthew O; Niemeyer, Glenn P; Vaglenov, Alex et al. (2013) Decreased hematopoietic progenitor cell mobilization in pearl mice. Exp Hematol 41:848-57