Effects of N‐oleoylethanolamide on Lymphoblasts Deficient in Tafazzin

Abstract

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in the TAZ gene that encodes for the cardiolipin remodelling enzyme, Tafazzin. This syndrome is characterized by cardiac and skeletal myopathies, as well as immunological deficits that cause significant morbidity and mortality. Study of the molecular pathology of BTHS thus provides insight into the role of cardiolipin and mitochondria in cellular physiology, while addressing a clinically relevant disease. Current treatment options for BTHS are limited, but a recent trial suggests that PPARα activation may be therapeutic. The objective of this study was to quantitatively examine the growth properties and mitochondrial physiology of B-lymphoblasts derived from five donors with BTHS, as well as effects of treatment with oleoylethanolamide (OEA), a bioactive N-acylethanolamine. Cyclic neutropenia is a well-recognized aspect of the pathology of BTHS. We hypothesized that deficits in lymphoblast proliferation would also be evident, and that OEA, which activates receptors including PPARα, would demonstrate therapeutic benefit. One million B-lymphoblasts derived from BTHS donors and age- and sex-matched healthy controls were seeded on day 0 and counted daily for 4 days. On day 4, there were 27.7 ± 4.6% fewer BTHS B-lymphoblasts than control cells (P<0.05), indicating that lymphopenia is likely also a component of BTHS. Treatment of B-lymphoblasts with 1 μM OEA caused no significant effect on control cell numbers, but significantly improved deficits in each of the five BTHS lymphoblast lines, raising the mean cell number on day 4 from 3.74 × 106 to 4.25 × 106 cells, although a full rescue of growth was not achieved. Analysis of electron micrographs of samples from two distinct BTHS B-lymphoblast donors indicated that cells had larger mitochondria as compared with healthy controls (mean of 0.67 ± 0.07 µM2 versus 0.27 ± 0.16 µM2, respectively). OEA decreased mitochondrial size by 53.0 ± 10.9%, while increasing the total mitochondrial number per cell by 92.6 ± 58.6%. OEA reduced the elevated expression of the enzyme CDS1 in BTHS B-lymphoblasts to healthy control levels, suggesting improved cardiolipin levels may play a role. However, an initial donor pair comparison did not show an effect of OEA on cardiolipin content, suggesting other mechanisms should be explored. Higher immunodetectable levels of OPA1, a biomarker of mitochondrial fusion, were found in lymphoblasts from children with BTHS, and were attenuated by OEA. Results from this study newly identify lymphopenia as a probable immunological deficit in BTHS, and suggest that therapies targeting aberrant fission/fusion of the mitochondrial network, rather than cardiolipin content, per se, and may have clinical utility for treatment of this disease.