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Neutropenia in Barth Syndrome: Endoplasmic reticulum stress and an increased sensitivity to apoptosis

Barth syndrome (BTHS) is an X-linked recessive disorder characterized by loss-of-function mutations in tafazzin (TAZ). Tafazzin is an enzyme that processes cardiolipin, a phospholipid found within the inner mitochondrial membrane; the lack of tafazzin activity results in a deficiency of unsaturated cardiolipins.  
In addition to problems with cardiomyopathy, patients with BTHS have a high childhood mortality rate due to neutropenia and life-threatening infections. The cellular and molecular mechanism of neutropenia in BTHS is unknown; here we examined the metabolism, function, and survival of neutrophils lacking TAZ in order to understand the link between TAZ-deficiency and neutropenia.  

We utilized a system of myeloid progenitor conditional immortalization established in our lab. In this system, an ER Hoxb8 fusion protein is used to conditionally immortalized granulocyte-monocyte progenitors from TAZ-KO and WT mouse fetal liver hematopoietic progenitors. These progenitors are then capable of unlimited culture and expansion ex vivo and can be differentiated upon demand to mature and functional neutrophils.   
TAZ-KO neutrophils showed no significant defects in phagocytosis, migration, and cytokine secretion. As previously demonstrated, TAZ deficiency in neutrophils significantly decreased mitochondrial oxygen consumption and intracellular ATP production rate. Lipidiomics analysis indicated that the loss of tafazzin function led to the expected reduction in unsaturated cardiolipins as well as a dysregulation of cholesterol, fatty acids, and other phospholipids. Consistent with these findings, gene expression analysis indicated that transcripts regulating lipid metabolism, steroid biosynthesis and AMPK signaling pathways were upregulated in TAZ-KO cells.

The TAZ-KO progenitors were more sensitive to apoptotic stress as triggered by ABT 199, an inhibitor of Bcl2. Moreover, the TAZ KO progenitors exhibited higher expression of genes such as CHOP (DDIT3) and ATF4, suggestive of endoplasmic reticulum (ER) stress. This increased expression of CHOP and ATF4, as well as other ER stress markers, was confirmed at the protein level by intracellular flow cytometry. 
To confirm these findings in vivo, wild-type recipient mice were transplanted with fetal liver hematopoietic progenitors from either wild-type or TAZ-KO donor embryos (E16). Twenty weeks later, these mice were challenged with thapsigargin, a chemical inducer of ER stress, and a heightened sensitivity to thapsigargin was demonstrated in the tafazzin deficient peripheral blood neutrophils. 
Overall, these findings point towards a mechanism of ER stress and apoptosis underlying the phenotype of neutropenia in patients with Barth syndrome. 

Jihee Sohn1, Thomas J. Brouse1, Suya Wang2, William Pu2 and David B. Sykes1

  1. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 
  2. Department of Cardiology, Boston Children’s Hospital, Boston, MA

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