Lipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria

Abstract

The objective of this study was to characterize the lipidome and electron transport chain activities in purified non-synaptic (NS) and synaptic (Syn) mitochondria from C57BL/6J mouse cerebral cortex. Contamination from subcellular membranes, especially myelin, has hindered past attempts to accurately characterize the lipid composition of brain mitochondria. An improved Ficoll and sucrose discontinuous gradient method was employed that yielded highly enriched mitochondrial populations free of myelin contamination. The activities of Complexes I, II, III, and II/III were lower in Syn than in NS mitochondria, while Complexes I/III and IV activities were similar in both populations. Shotgun lipidomics showed that levels of cardiolipin (Ptd2Gro) were lower, whereas levels of ceramide and phosphatidylserine were higher in Syn than in NS mitochondria. Coenzyme Q9 and Q10 was also lower in Syn than in NS mitochondria. Gangliosides, phosphatidic acid, sulfatides, and cerebrosides were undetectable in brain mitochondria. The distribution of Ptd2Gro molecular species was similar in both populations and formed a unique pattern, consisting of seven major molecular species groups, when arranged according to mass to charge ratios. Remodeling involving choline and ethanolamine phosphoglycerides could explain Ptd2Gro heterogeneity. NS and Syn mitochondrial lipidomic heterogeneity could influence energy metabolism, which may contribute to metabolic compartmentation of the brain.

Dynamic simulation of cardiolipin remodeling: greasing the wheels for an interpretative approach to lipidomics

“…3B ). For example, a similar broad distribution of cardiolipin molecular species was observed between day 5 and 15 for normal mouse cardiac development (data not shown) as well as in mature mouse brain ( 16,26 ). It is interesting that this broad distribution of cardiolipin molecular species is also observed in various disease states, culture conditions, and transgenic models of enzymes that catalyze cardiolipin remodeling ( 23,(41)(42)(43)(44)(45).…”

“…Although that approach demonstrated novel insight into potential mechanisms governing acyl selectivity, it did not directly address the importance of kinetics in the generation of cardiolipin acyl chain distributions. In addition, that approach only addressed distributions that are relegated by tetralinoleic abundance of cardiolipin; however, brain cardiolipin does not conform to that model ( 16,26 ). Moreover, alterations in free energies are complex functions of enthalpy and entropy driven by altering molecular membrane symmetry and the order/disorder at the membrane interface.…”

“…The murine mitochondrial lipidome from brain, liver, lung, and heart was determined using MDMS-SL as previously described ( 26 ). Briefl y, a 1 mg aliquot of mitochondrial protein was transferred to a disposable borosilicate glass tube (16 × …”

“…Brain cortex, heart, liver, and lung of mice were harvested immediately after perfusion. Nonsynaptic brain mitochondria were isolated as previously described in detail using discontinuous Ficoll and sucrose gradients ( 25,26 ). Myocardial, hepatic, and lung mitochondria were isolated as previously described ( 27 ).…”

Myocardial Regulation of Lipidomic Flux by Cardiolipin Synthase

“…Western blot analyses of isolated mitochondria were performed as described previously (34). Polyclonal antibodies for Western blots were obtained from Protein Tech human CLS (14845-1), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (CDS2 (P-14) and tafazzin (JK-2)), Aviva Systems Biology (PGS1-ARP48895), MLCL AT (a generous gift from Dr. Grant Hatch, University of Manitoba), and SigmaAldrich (VDAC-V2139).…”