Disruption of Mitochondrial Phosphatase Ptpmt1 Induces Bioenergetic Stress and Differentiation Block in Hematopoietic Stem Cells

Abstract 857

Bioenergetic coordination of hematopoietic stem cell (HSC) maintenance and lineage differentiation remains largely unknown. The hypoxic microenvironment in stem cell niches limits mitochondrial aerobic metabolism (oxidative phosphorylation) in HSCs, which preserves this essential cell reservoir from oxidative damage by attenuating the production of reactive oxygen species (ROS), a byproduct of oxidative phosphorylation. However, the mitochondrial metabolic mechanisms regulating HSC differentiation in concert with other regulatory networks are poorly defined. Ptpmt1, a PTEN-like phosphatidylinositol phosphate (PIP) phosphatase exclusively localized in the mitochondria, is highly expressed in HSCs relative to lineage progenitor and mature cells. To dissect the role of Ptpmt1 in hematopoietic cell development, a Ptpmt1 conditional allele was created. Depletion of Ptpmt1 in inducible (Ptpmt1^fl/fl/Mx1-Cre⁺) or hematopoietic cell-specific (Ptpmt1^fl/fl/Vav1-Cre⁺) knockout mice resulted in fatal anemia and pan cytopenia. Ptpmt1 knockout bone marrow cells failed to reconstitute the hematopoietic system in competitive and non-competitive transplantation settings. Surprisingly, the HSC pool was increased by ∼40-fold in Ptpmt1 knockout mice. The knockout HSCs were delayed in the G₁ phase in the cell cycle due to marked upregulation of cyclin dependent kinase inhibitors p21 and p57. Reintroduction of wild-type, but not catalytically-deficient Ptpmt1 or truncated Ptpmt1 lacking mitochondrial localization signal, restored repopulating capabilities of Ptpmt1 knockout HSCs in transplants, suggesting that Ptpmt1 functions in a catalytically-dependent fashion during stem cell differentiation and that the mitochondrial localization is required for its function. Depletion of Ptpmt1 did not affect the survival of stem cells and ROS levels were unchanged. However, mitochondrial aerobic metabolism decreased while glycolysis was enhanced in Ptpmt1-depleted HSCs. As a result, AMP-activated kinase (AMPK), an energy stress sensor, was highly activated in these cells. Furthermore, PIP substrates of Ptpmt1 directly enhanced fatty acid-induced activation of mitochondrial uncoupling protein 2 (UCP2). Total proton conductance of UCP2-reconstituted plannar bilayer membranes was significantly increased with the addition of these PIPs. Intriguingly, depletion of Ptpmt1 from either myeloid (Ptpmt1^fl/fl/LysM-Cre⁺), T lymphoid (Ptpmt1^fl/fl/Lck-Cre⁺), or B lymphoid (Ptpmt1^fl/fl/CD19-Cre⁺) progenitors did not cause any defects in lineage-specific knockout mice, although these cells displayed metabolic alterations similar to those in Ptpmt1-depleted stem cells. Taken together, this study establishes the crucial role of Ptpmt1 phosphatase in hematopoiesis and suggests the existence of a stem cell differentiation checkpoint regulated by mitochondrial bioenergetic metabolism.