Valerio Leoni (Presenter)
Lab of Clinical Chemistry, Hospital of Varese
Authorship: Valerio Leoni (1), Andrea Nauti (1), Caccia Claudio (2)
(1): Laboratory of Clinical Chemistry, Hospital of Varese, ASST-Settelaghi, Varese, Italy; (2) Laboratory of Clinical Pathology, Foundation IRCCS Carlo Besta, Milano, Italy
Mitochondrial alterations are associated to tricarboxylic acid cycle and OXPHOS dysfunction which result into a reduced production of ATP, NADPH and Acetly-CoA. These dysfunctions induce changes to the lipid composition of the cellular membranes as consequence of an anabolic impairment. Isitope-dilution mass spectrometry metabolomics allo to investigate the changes in the cellular and tissue lipid profile relating functional to genetic and metabolomics evidences. It is likely that this mechanism might contribute to the process of neurodegeneration.
Introduction: Cholesterol and fatty acid (FAs) esters as phospholipids and sphingolipids are structural elements of cellular membrane. In the central nervous system, cholesterol is involved in
synaptogenesis, maintenance, turnover, stabilisation and restore of synapses. Within the brain,
about 70% of cholesterol is present in myelin sheath formed by sections of oligodendrocyte plasma membrane repeatedly wrapped around an axon, with the extrusion of virtually all of the cytoplasm. Changes in lipid composition of plasma and brain were reported in neurological disorders (NDs) such as Parkinson disease, Huntington Disease (HD) and Multiple Sclerosis, and were proposed as predictors of disease progression. Abnormalities of the plasma phospholipids and of the cholesterol metabolism biomarkers were also reported in aging, obesity, T2D, metabolic syndrome, dyslipidemia, which are, altogether, risk factors for AD and NDs. Alterations of the lipid composition of neuronal membranes enhance formation of Aβ. Lipid metabolism is also an emerging therapeutic target in NDs.
Mitochondria with tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) are the
production site of energy and substrates for cellular lipid biosynthesis. FAs and cholesterol are
synthesized from acetyl-CoA originating from mitochondrial citric acid transported in the cytosol and cleaved by ATP-citrate lyase. Abnormalities in mitochondrial dynamics and functions were observed in NDs, aging and age-related diseases.
Materials and Methods: Sterols, oxysterols, fatty acids and metabolic organic acid were measured by isotope dilution gas chromatography-mass spectrometry (Perkin Elmer Clarus 600) after ethanolic saponification, liquid to liquid and cartridge separation and TMS-derivatisation in cultured cells exposed to oxysterols, rifampicine, metformin with a pro-apoptotic and autophagic stimulation. Crystal violet test and phase contrast microscopy to evaluate cell growth and the loss of cell adhesion; flow cytometry and confocal microscopy with an antibody raised against the peroxisomal membrane protein Abcd3 to evaluate the peroxisomal mass; western blot analyses to precise the impact of 7KC on the mitochondrial status (oxidative phosphorylation (OXPHOS antibodies); mitochondrial proliferation/quantity of mitochondria (situin-1 level and activity, PGC1-α level and phosphorylation, AMPK level and phosphorylation, MTC02 level). The induction of oxiapoptophagy (oxidation + apoptosis + autophagy) was determined by flow cytometry by staining with dihydroethidine allowing quantification of reactive oxygen species overproduction, and by western blotting (quantification of cleaved caspase-3; activation of LC3-I into LC3-II and quantification of the ratio LC3-II/LC3-I).
Results: In presence of pro-apoptotic stimulation, morphologic and functional abnormalities of
mitochondria were observed. The transmembrane mitochondrial potential (Δψm) was reduced, the cellular amount of lactate was higher while pyruvate, citrate, fumarate, succinate (TCA
intermediates) were significantly reduced, suggesting that an impairment of mitochondrial
respiratory function lead to an increase of lactate production and a reduced amount of ATP, NAD and Acetyl-CoA available for the anabolic pathways. Sterol precursors (lathosterol, lanosterol and
desmosterol) and satured and unsatured long chain fatty acids (C16 - C18, structural elements of
membrane phospholipids) were reduced. Changes in oxysterol composition were observed. In brain samples were observed a significant reduction of sterol precursors, 24OHC and 27OHC and changes in fatty acids profile.
Conclusions & Discussion
Conclusions: Mitochondrial alterations are associated to TCA and OXPHOS dysfunction which result into a reduced production of ATP, NADPH and Acetly-CoA. These dysfunctions are able to induce changes to the lipid composition of the cellular membranes as consequence of an anabolic
impairment. It is likely that this mechanism might contribute to the process of neurodegeneration.
References & Acknowledgements:
IP Royalty: no
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