Investigation of the therapeutic potential of a new phytoceutical for lymphopenia in Barth Syndrome

Abstract

Background: Barth syndrome (BTHS) is a debilitating X-linked genetic disorder caused by mutations in the gene that encodes for Tafazzin, an enzyme important for cardiolipin (CL) remodeling. This disorder impairs the ability to modify fatty acyl chains on cardiolipin after de novo synthesis, resulting in decreased cardiolipin levels and alterations in cardiolipin fatty acyl composition. Given that CL is crucial for mitochondrial processes, TAFAZZIN mutations lead to abnormal mitochondrial ultrastructure, impaired dynamics, and decreased electron transport chain capacity. As a result, tissues with high energy demands, including the heart, skeletal muscle, and immune system, are most affected by BTHS. While BTHS research has traditionally focused on innate immune dysfunction, recent studies highlight potential adaptive immune impairments. Data from my MSc research demonstrated that transformed B-lymphoblasts derived from individuals with BTHS exhibit significantly reduced growth compared to sex- and age-matched healthy controls. This deficit was partially restored with the endocannabinoid N-oleoylethanolamide (OEA). However, while OEA showed initial promise, it only partially rescued the impaired clonal expansion of these cell lines. In the pursuit of compounds with potentially better efficacy and safety profiles, further investigations turned to a natural phytochemical known for its antioxidant and mitochondrial-modulating properties, which we refer to as Compound X.

Aims and Approach: This thesis aimed to evaluate the therapeutic potential of Compound X in treating BTHS-associated lymphopenia. To achieve this, several objectives were examined by comparing responses in both BTHS and healthy B-lymphoblasts. Specifically, we: (1) assessed the effects of Compound X on cell growth using manual cell counting with a hemocytometer; (2) analyzed changes in CL content and composition following Compound X treatment via thin-layer chromatography and gas chromatography (TLC-GC); (3) evaluated the expression of genes involved in CL biosynthesis using RT-qPCR; (4) examined the impact of Compound X on ETC protein subunit levels using immunoblotting; and (5) investigated alterations in mitochondrial ultrastructure and morphology in response to Compound X treatment through transmission electron microscopy (TEM) and immunoblotting of fission and fusion mediators.

Results: In the first study, Compound X treatment fully restored the impaired cell expansion of B-lymphoblasts derived from five distinct BTHS donors, bringing the cell number to near levels of healthy controls. This growth improvement was accompanied with significant increases in the maximal coupled state III respiration in BTHS B-lymphoblasts, in which all five tested BTHS donors had increased mitochondrial membrane potential following Compound X treatment. Further analysis revealed that Compound X treatment restored total CL content of BTHS B-lymphoblasts to healthy levels, but did not significantly alter the relative proportions of the major CL fatty acyl species. At the protein level, Compound X treatment reversed the decreased expression of major ETC subunits, including succinate dehydrogenase subunit A (SDHA), cytochrome c oxidase subunit 1 (COX1), and cytochrome c oxidase subunit IV (COXIV). TEM analysis of three distinct BTHS donor cell lines showed that BTHS lymphoblasts had abnormal cristae patterns with smaller total cristae lengths and enlarged mitochondria compared to healthy controls. While Compound X treatment did not significantly increase the length of cristae, it significantly reduced the mitochondrial enlargement in BTHS B-lymphoblasts. Lastly, immunoblotting of fusion mediators revealed elevated OPA1 levels in BTHS lymphoblasts, while analysis of the fission mediator DRP1 revealed elevated phosphorylation of its inhibitory site at Serine 637, both of which were significantly attenuated by Compound X treatment, suggesting a potential role for this compound in restoring mitochondrial dynamics in BTHS B-lymphoblasts.

Conclusion: These findings demonstrate for the first time that Compound X effectively rescued the observed impairments in B-lymphoblast cell expansion in BTHS and partially restored BTHS B-lymphoblast mitochondrial function and morphology. However, additional experimentation in both animal models and humans are needed to further validate the therapeutic efficacy of Compound X in BTHS lymphopenia.