The Cellular and Molecular Mechanisms of Neutropenia in Barth Syndrome

Abstract 1105

Barth syndrome (BTHS) is a rare, X-linked recessive disease characterized by progressive cardiomyopathy and neutropenia with life-threatening bacterial infections. BTHS is attributable to loss-of-function mutations in the tafazzin gene, TAZ, a phospholipid acyltransferase localized to the mitochondrial membrane and involved in remodeling cardiolipin. BTHS children exhibit reduced blood levels of total cardiolipin; however, the molecular and cellular mechanisms of BTHS remain largely unknown.

We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrated a significant down-regulation in the TAZ gene expression, mimicking naturally-occurring truncation mutations. FACS analyses of cells with TAZ-specific but not scrambled shRNAs demonstrated nearly two-fold increase in proportion of annexin-V positive cells and significantly increased dissipation of mitochondrial membrane potential (MMP) as determined by DIOC6-staining. Similar pro-apoptotic effects were observed in U937 myeloid progenitors, but not in Jurkat or Ramos human lymphoid cell lines. Further studies revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knock-down. Treatment with caspase-specific inhibitor zVAD-fmk reduced apoptosis to near-normal levels. FACS analysis utilizing cryopreserved bone marrow cells from a BTHS patient showed evidence of accelerated apoptosis compared to corresponding cells from a healthy volunteer. Importantly, human myeloid progenitor cells and cardiomyoblasts/cardiomyocytes depend heavily on mitochondria, which may explain why the loss of mitochondrial protein tafazzin has more severe effects on these specific tissues in BTHS resulting in neutropenia and cardiomyopathy.

These data suggest that neutropenia in BTHS patients is attributable to increased dissipation of MMP, aberrant release of cytochrome c, activation of caspase-3 and accelerated apoptosis, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors. Further studies should evaluate the TAZ–regulated mitochondrial apoptosis pathway using primary cells from BTHS patients.