Promotion of plasmalogen biosynthesis reverse lipid changes in a Barth Syndrome cell model
Introduction
Barth Syndrome (BTHS) is an X-linked recessive disease caused by mutations in the gene that encodes tafazzin [1,2]. Tafazzin is a lysophospholipid-phospholipid transacylase involved in the last step of cardiolipin (CL) biosynthesis [3,4]. In BTHS loss-of-function of tafazzin leads to a lower content of CL as well as more heterogeneous molecular species of this lipid and accumulation of monolysocardiolipin (MLCL), which are considered the hallmark of BTHS [[5], [6], [7]]. In eukaryotes, CL is a mitochondria-specific lipid, which has been reported to play crucial roles in the structure and function of mitochondria [8]. Hence, it is not unexpected that a major focus of research in this disease has been to understand the molecular events resulting from changes in both the quantities and the molecular species of CL on mitochondrial structure and function.
In BTHS mitochondrial morphology is aberrant, presenting swollen and enlarged mitochondria with malformed cristae structures, which have lower surface area and appear round and disconnected from the peripheral membrane, instead of parallel cristae that often extend across the entire organelle observed in controls [9]. In addition, CL also plays a role in keeping the integrity of the inner mitochondrial membrane (IMM) and alterations that compromise it would result in dissipation of the proton gradient that drives ADP phosphorylation. Indeed, BTHS mitochondria exhibit increased proton leakage, decreased membrane potential, and a decreased respiratory coupling index [[9], [10], [11], [12]]. Moreover, in Barth specimens, changes in CL lead to destabilization of mitochondrial super complexes (SC) [9,13,14]. Likewise, assembly of mitochondrial SC induces CL remodeling and, as a consequence, its stabilization [15]. Mitochondria are key players in cellular redox metabolism and disruption of mitochondrial SC is known to result in increased production of reactive oxygen species (ROS) [16,17]. Indeed, in several BTHS specimens elevated ROS production has been reported [9,12,18,19]. Cells from BTHS patients usually display increased mitochondria biogenesis as indicated by the rise in the AMP/ATP ratio and mitochondria copy number when compared to controls, which is proposed to occur for cellular compensation due to the impaired mitochondria [9,11].
While the alterations in CL associated with BTHS are well-established, currently there has been an increased awareness of more widespread changes in the lipid composition of cells depleted of tafazzin function. Of note, it is the recent identification of a decrease in the plasmalogen levels in organs obtained from tafazzin knockdown mice as well as blood cells derived from BTHS patients, which clearly indicate that plasmalogen loss is associated with BTHS [20,21].
Plasmalogens are members of a subclass of glycerophospholipids, which differ from their diacylglycerophospholipid counterpart by having a fatty alcohol linked via a vinyl-ether bond at the sn-1 position of the glycerol moiety rather than an esterified fatty acid. Plasmalogens comprise up to 20% of the total phospholipid mass in humans and are constituents of the plasma membrane as well as intracellular membranes (nucleus, endoplasmic reticulum, post-Golgi network, mitochondria) [22]. Plasmenylethanolamine (PE-Pls) is the predominant plasmalogen found in most of human cells, except in cardiac and skeletal muscles and mature spermatozoa where high proportions of both PE-Pls and plasmenylcholine are found [22]. Although the physiological role of plasmalogen remains elusive, several studies had shed light on some of their physical, chemical, and biological functions. For instance, it has been reported that the vinyl-ether bond at the sn-1 position of the glycerol moiety brings the aliphatic chains closer together in comparison to their diacyl counterparts [23,24]. This leads to more packed membranes with decreased fluidity and increased order, while more easily promoting the formation of non-bilayer phases. In addition, the vinyl-ether bond is oxidation-labile rendering plasmalogens the ability to scavenger ROS, which led to the proposition they could act as a protection against the oxidation of polyunsaturated fatty acids (PUFAs) and other oxidation prone lipids [[25], [26], [27]]. Moreover, plasmalogens are believed to act as reservoirs of second messengers as they are, usually, enriched with PUFAs at the sn-2 position of the glycerol moiety, which are biologically active lipid mediators released upon plasmalogen hydrolyses by PLA2 [22].
A decrease in the steady-state levels of plasmalogens is associated with an impaired biosynthesis or an enhanced degradation. Currently the mechanism of plasmalogen depletion in BTHS at the molecular level is not known. However, in several diseases characterized by depletion in plasmalogens, promotion of their synthesis has been shown as a successful strategy to alleviate/treat those conditions [22]. Furthermore, disruption of plasmalogen biosynthesis impaired mitochondrial dynamics, which could be rescued upon promotion of plasmalogen biosynthesis [28]. While restoring plasmalogen levels is relatively straightforward to accomplish, previous attempts to increase CL levels were not successful by direct administration of this lipid in vivo [29]. Hence, it was hypothesized that by promoting plasmalogen biosynthesis it would be possible to restore to normal the levels of this lipid in Barth specimens and, eventually, normalize the lipidome and mitochondria function. To do so lymphoblasts derived from BTHS patients were used and compared with controls. Cells were administered with plasmalogen precursors and the lipid profile was characterized by high resolution 31P NMR phospholipidomic analysis and mass spectrometry and compared with cells without the administration of plasmalogen precursors. The effect of the treatment on cell viability, mitochondria biogenesis, and mitochondria membrane potential were also evaluated.
It is shown that the promotion of plasmalogen biosynthesis could restore the low levels of PE-Pls in this BTHS cell model to levels comparable to the control. In addition, the data showed a compensatory effect of the diacyl counterpart lipid upon plasmalogen precursors administration; that is, a decrease in the steady-state levels of diacyl-PE. Noteworthy, promotion of plasmalogen biosynthesis led to a significant increase in the levels of CL in BTHS lymphoblasts, while no changes were observed in the MLCL levels. Promotion of plasmalogen biosynthesis did not impact cell viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential to levels comparable to controls. Overall, the results highlight the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and suggest that a diet supplemented with plasmalogen precursors could be beneficial for BTHS patients.
Section snippets
Materials
Sodium dodecyl sulfate (SDS) was from Bioshop (Canada). 2-ethanesulfonic acid (MES), ethanolamine, CsOH, butylated hydroxytoluene (BHT), ethylenedinitrilo-tetraacetic acid (EDTA), bovine serum albumin (BSA), carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP), methanol (HPLC grade), and chloroform (HPLC grade) were from Sigma-Aldrich (St. Louis, MO). D2O (99.9 atom %D) was from Cambridge Isotope Laboratories (Tewksbury, MA). Roswell Park Memorial Institute (RPMI) 1640 Medium was from
Effect of the promotion of plasmalogen biosynthesis on the phospholipidome of BTHS lymphoblasts
To reverse the low levels of plasmalogens found in Barth specimens, a strategy based on the promotion of their biosynthesis was used. The effects of the promotion of plasmalogen biosynthesis were studied using lymphoblasts derived from BTHS patients and compared with controls. The cells from Barth patients have all the characteristics of cells from patients with this Syndrome, including the decrease in the levels of plasmalogens [9,11,21]. Plasmalogen biosynthesis begins in peroxisomes and is
Discussion
Currently, there is no specific treatment for BTHS. Contrary to the well-established alterations in CL content and molecular species in BTHS, the recent identification of a marked decrease in another class of lipids, namely plasmalogens, in BTHS has opened a new possibility, up to now not considered, as a potential strategy to alleviate/treat this condition, which is through the promotion of plasmalogen biosynthesis [20,21]. It has been shown in other systems that restoring plasmalogen levels
Conclusions
To sum up, the present study showed that it is possible to restore the low levels of plasmalogen in a BTHS cell model upon promotion of its biosynthesis. Importantly, the administration of plasmalogen precursors did not impact on the cell viability, while it increased to some extent the CL levels as well as restored mitochondrial properties. Until the association of plasmalogen loss with BTHS such strategy could not have been considered and the results presented here suggest that a diet
Declaration of competing interest
The authors declare that they have no conflicts of interest with the content of this article.
Acknowledgements
We appreciate the technical support of Hong Liang (McMaster University) with flow cytometry measurements and Omar El-Halfawy (McMaster University) with real-time PCR measurements. We thank Shamin Fernando for his help with lipid extraction for LC-MS analysis.
Funding
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant RGPIN-2018-05585 to R.M.E and the National Science Foundation grant MCB1817468 to G.E.A.-G. and the Research Foundation for the State University of New York (J. Solo funds) to G.E.A.-G..
Author contributions
J.C.B. Jr. performed cell work, cellular ATP content quantification, mitochondria copy number quantification, and high resolution 31P NMR measurements. D.L. performed mass spectrometry measurements under the supervision of G.E.A.-G. J.C.B. Jr. and R.M.E. designed the experiments, analyzed the data, and wrote the manuscript.
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