Interrogation of drug effects on the lipid composition of single cells and Drosophila brain using ToF-SIMS imaging

Doctoral thesis, 2020

Lipids are essential for all living organisms on Earth. The most important function of lipids is that they act as the building blocks of cellular membranes. Lipids consist of polar head groups and non-polar tail groups and assemble into bilayer structures to create cell and organelle membranes. The plasma membrane of a cell provides a barrier which segregates cellular internal constituents from the external environment. In addition to acting as a barrier, membrane lipids are involved in many cellular processes including membrane trafficking, signal transduction, fission and fusion. Therefore, various conditions in the central nervous system involving lipid deficiencies can lead to function deficit. There are several drugs that induce the dysregulation of lipid metabolism linked to the impairment or enhancement of cognitive function. Hence, I have studied the lipid alterations in brain induced by drugs with regards to their effects on cognitive processes: cognitive impairing drugs (cocaine and zinc deficiency) and cognitive enhancing drugs (methylphenidate and fatty acids). Much work has been done to investigate the link between lipid metabolisms and these drugs. A powerful technique for lipid analysis is mass spectrometry imaging (MSI). MSI is a surface sensitive method which enables label-free detection of molecules in complex biological systems. In addition, MSI provides the relative composition as well as allows imaging of intact species with high spatial resolution in single experiments. One of the most common MSI techniques is time-of-flight secondary ion mass spectrometry (ToF-SIMS), which achieves high spatial resolution using a focused ion beam to eject and ionize molecules in the sample surface. Recently, gas cluster ion beams have been introduced to reduce the chemical damage during sampling of surfaces and to achieve enhancement of lipid signals. In our studies, ToF-SIMS has been applied to lipids in the membranes of cells and Drosophila melanogaster brain to get a better understanding about the effect of drugs in lipid mechanisms related to neuronal signal transmission.

The papers included in this thesis describe the application of ToF-SIMS in biological samples to reveal the alterations of lipids after drug treatments. In paper I, the alterations in lipid distribution and composition induced by cocaine and methylphenidate, which cause the impairment and enhancement in cognitive performance respectively, were investigated. ToF-SIMS data were used to show that cocaine and methylphenidate have opposite effects on the relative levels of lipids in the central fly brain. To enhance our understanding about the lipid mechanisms, in paper II, I used stable deuterium-labeled omega-3 and -6 fatty acids as lipid precursors to analyze the synthesis and transportation of lipids into the plasma membrane of PC12 cells. The use of isotope-labeled fatty acids provided a tool to track the lipid turn-over as well as to measure their relative amounts. Paper III continued the work done in paper I, where experiments were performed to investigate the recovery of lipids after cocaine removal. In addition, the cognitive-enhancing drug, methylphenidate, was used to treat cocaine removal from flies to investigate the reversal of lipid changes in the brain caused by repeated-cocaine exposure. Zinc deficiency in the diet, which causes a decrease in cognitive function, was also studied in fly brain. ToF-SIMS data obtained reveal that the lipid types that change are similar to those when treated with cocaine as seen in paper IV.

ToF-SIMS opens a new approach to visualize and relatively quantify phospholipids in biological tissues and cells. In the biological model systems studied here, cognition-affecting drugs show that alterations in the distribution and composition of specific lipids is altered differently based on whether the drug enhances versus diminishes cognition. These results provide new possible targets for lipid-modifying therapies to improve the cognitive decline in drug abuse and diseases.