Elucidating the Role of the Endocytic Protein Ap2a2 in Haematopoiesis

The rare subpopulation of quiescent HSCs exists in limited numbers but upon in vivo requirement have the highest proliferative potency. Self-renewal via asymmetrical cell division (ACD) is vital for maintenance of HSC numbers and perturbation of this process can lead to an impaired hematopoietic system, with subsequent blood diseases such as acute leukemia and/or bone marrow failure. How and which HSCs are programmed for ACD remain central and unanswered questions. We have demonstrated that the endocytic protein, Ap2a2 distinguishes an asymmetrical from a symmetrical cell division during HSC mitoses, and when overexpressed enhances mouse HSC activity.Ap2a2 is a component of the Adaptor protein 2 complex that functions in clathrin-dependent endocytosis of receptors and nutrient cargo.

The tetracycline-induceable H2B-GFP mouse (Foudi A. et al, Nature Biotech 2009) has revealed functional heterogeneity within the CD150⁺48^- Lin^-Sca⁺Kit⁺(LSK) HSC population, such that the GFP^high subpopulation within the CD150⁺48^- LSK is the true Long-Term repopulating HSCs (GFP^high LT-HSCs). Using this mouse, we have shown that Ap2a2 is not only more highly expressed in the GFP^high LT-HSCs but that Ap2a2 overexpression increases the fraction of this specific subpopulation from 20% to 60% at plus 20 weeks post-transplantation. To identify novel genes and pathways driving LT-HSC function and self-renewal, we are currently performing comparative gene expression analyses from the subpopulation of Ap2a2-transduced against control, vector-transduced GFP^highCD150+48-LSK LT-HSCs.

To further investigate Ap2a2 in haematopoiesis, we have constructed an Ap2a2 conditional knockout mouse (Ap2a2 cKO) line, which has a LacZ reporter expressed from the endogenous Ap2a2 promoter. Our initial expression analyses with tissue LacZ staining shows relative increased staining in subpopulations of bone marrow cells and using a β-galactosidase (β-gal) flow cytometry method co-stained with CD150+48-LSK, have identified β-gal^high and β-gal^low subpopulations. This suggests Ap2a2 expression may be an independent marker of LT-HSCs, which we are functionally assessing via comparative LSK -β-gal^high versus -β-gal^low versus -β-gal^negative transplantation assays. The specific function of Ap2a2 in developmental (Vav-Cre) and adult (Mx-Cre) haematopoiesis is being investigated by respective tissue specific-Cre matings with the Ap2a2^fl/flmice.

Mechanistically, a recent publication (Ito K. et al, Nature Med 2012) identified the importance of PPAR-δ/β-fatty acid oxidation regulation for both maintenance and ACD ofHSCs. Ap2a2 is a target of PPAR-α (Buroker N. et al Protein J 2012). Our hypothesis states that the role of Ap2a2 in LT-HSCs involves the lipid metabolic pathway, which would uniquely link endocytosis to metabolism. To this end, using our Ap2a2-LacZ reporter mouse, we have shown intense and specific LacZ expression in adipocytes of the bone marrow and other lipid containing organs. In addition, perturbation of Ap2a2 results in altered lipid droplet formation and impaired triacylgylceride storage. We are currently investigating the oxidative phosphorylation, anaerobic glycolysis and fatty acid metabolic effects in the context of altered Ap2a2 expression.