Lipid rafts in brain cellsfrom enzymatic activity to lipidomic assays in a novel printed rafts platform

Resum

Changes in cellular homeostasis, such as oxidative stress, can trigger neurodegeneration, producing cellular pathological changes such as lipid peroxidation, oxidative modification of proteins, and DNA damage. Reactive Oxygen Species (ROS), key deregulators of cellular stability, can be produced by different oxidoreductase enzymes and by the mitochondrial electron transport chain (mETC). Many of these enzymes are located in liquid-ordered membrane domains, so-called lipid rafts, dynamic platforms that participate in various signaling pathways. Moreover, changes in the lipidic environment of membrane proteins can lead to important functional disruptions. Lipidomic and enzymatic activity assays have been successfully performed in printed cell membrane homogenates (cell membrane microarrays, CMMAs). Based on that technology and the importance of liquid-ordered subdomains, we have developed a printed lipid raft and non-raft platform (raft membranes microarrays, RMMAs), with preserved native protein structure and lipidic environment. To evaluate the differences over lipidic environment in raft and non-raft subdomains in control, metabolic and oxidative stress conditions MALDI-MS assay was performed on RMMAs. Raft and non-raft subdomains presented a distinguishable fingerprint in every condition in both cell types (astrocytes and neurons). Distinguishable lipid fingerprints were also observed comparing raft subdomains obtained from cells in control, metabolic and oxidative stress situations. In the same way, raft and non-raft domains from control astrocytes and neurons are also distinguishable. Therefore, the lipidomic data obtained with this methodology can be used as a classification tool for samples of raft and non-raft subdomains with different treatments. As the lipidic environment is a key aspect for proper physiological enzymatic activity, changes over lipidome can lead to differences between enzymatic activities. To evaluate the differences in enzymatic activities inside the raft domains between stressed and non-stressed astrocytes, NADH oxidoreductase, GADPH, and Cholinesterase activity assays were performed on RMMAs. Higher NADH oxidoreductase activity in raft domains from metabolically stressed astrocytes was observed, whereas no differences were observed in GAPDH and Acetylcholinesterase activities. By contrast, rafts from neuronal samples presented high activity of both GAPDH and Acetylcholinesterase. Furthermore, NADH and GAPDH activities revealed a positive correlation between them and specific phospholipids, but surprisingly not with sphingolipids, one of the main components of lipid rafts. Thus, these data show the close relationship between lipidic structure in liquid-ordered domains and enzymatic activities. The results presented in this Ph.D. Thesis reveal the importance of a proper lipidic environment in lipid raft domains, and the impact over enzymatic activities, in two major cell types of the nervous system. This Thesis demonstrates the suitability of this newly-developed technology to make high-throughput analysis of lipid environment-function relationships in printed RMMAs of neuronal and astrocyte membranes.