Lipid Tracing in Murine Brain Tissues

Author: Will Bowman

Astrocytes and oligodendrocytes in grey and white matter regions of the brain metabolize fatty acids

Authors: Kristina Hofmann, Rosalia Rodriguez-Rodriguez, Anne Gaebler, Núria Casals, Anja Scheller & Lars Kuerschne

 

Authors have made significant progress by developing four alkyne lipid tracer molecules from both saturated and unsaturated families that mimic natural lipids in structure and biological behavior (Tiele, C. et al., 2012). This system has allowed parallel lipid tracking and quantification by fluorescence microscopy and TLC techniques respectively (Hofmann, K. et al., 2014). These tracers are able to follow lipid synthesis into membranes and other purposes, except catabolism. Fatty acid oxidation transforms the energy rich lipids into usable energy and generates essential building blocks such as acetyl-CoA in a cell. Tracers designed by the authors are unable to follow such pathways however, and the catabolism pathways are therefore not investigated in this paper.

 

Lipid biosynthesis typically occurs throughout the body to generate lipids for use in membranes and other functions. In the brain however, it is not known how much of the cerebral cells types contribute to lipid production or the varying rates at which lipids are produced. It is known however, that white matter and grey matter in the brain have differing levels of poly-unsaturated fatty acids (PUFAs) and sphingolipids (O’Brien, 1965). White matter is dominated by macroglia such as oligodendrocytes and astrocytes, which are responsible for myelination of neuron axons within the brain. Myelin lipids are composed of about 80% C18 or less fatty acids and about 6% PUFAs (Simons and Nave, 2015). Grey matter is dominated by neurons, which possess elongated cell shape and large membrane to volume ratio. In neurons it has been found that lipids such as cholesterol are not generated within the neuron but are donated from surrounding macroglial cells in both grey and white matter (Pfrieger and Ungerer, 2011). Authors confirmed the ability of oligodendrocytes and astrocytes to be able to perform lipid biosynthesis using labeled alkyne oleate as a biomarker.

 

Poly-unsaturated fatty acids are critical to healthy brain function, and are actively transported across the blood-brain barrier using systems that stifle transport of fatty acids derived from PUFAs (Edmond, 2001; Zhang et al., 2017). These critical lipids are not synthesized within humans and are entirely sourced from our diets, such as fish oil. Therefore, PUFAs are entirely reliant on the intra-cerebral lipid transport systems within the brain to reach cells like neurons which are highly enriched in PUFAs.

 

Authors traced lipids through the brain through staining and microscopy and found labeled lipids in high concentrations in the medial habenula. This gray matter structure forms a central structure connecting to the forebrain of mice and is thought to integrate cognition with emotional and sensory processing. This function plays a role in rewarding and aversive effects of stimuli (Boulos et al., 2017). The habenula also regulates monoaminergic systems and is thought to contribute to memory and learning processes (Lecourtier and Kelly, 2007). It was found that there is a sharp distinction between the edges of the medial habenula and surrounding brain tissues in terms of lipid distribution, lipids did not spread out from within the medial habenula to surrounding tissues. Macroglial habenular cells were found to be involved in local lipid metabolism, and further work confirmed lipid targeting of macroglial cells.

 

Lipid distribution in the brain was determined through the use of four alkyne labeled fatty acids, analogues of palmitate, stearate, oleate, and linoleate were used. These tracers were added to brain slice cultures and allowed to equilibrate over time. Authors made use of fluorescence microscopy to examine lipid staining, and found considerable lipid staining in grey matter regions such as the medial habenula, as well as some moderate staining in thalamus and cortex (shown in the image above). White matter regions were likewise labeled and examined, and pronounced white matter staining was found in all regions tested, the corpus callosum, internal capsule, and fimbria hippocampi. This uptake was found to be contingent on active metabolism, as fixing cells before incubating with tracers did not result in appreciable staining.

 

Authors determined that cells labeled in the previous experiment resembled macroglial cells. To confirm this, the experiment was repeated on transgenic fluorescent reporter mice. These animals expressed fluorescent protein with promoters for both oligodendrocyte marker proteins or astrocyte proteins. This allowed authors to confirm the accumulation of tracer lipids in the macroglial cells previously observed.

 

From these results, authors have suggested that macroglial cells are involved in lipid metabolism and biosynthesis in the cerebrum in vivo. Of note, astrocytes were found to produce contain a higher relative content of neutral lipid triacylglycerol in contrast to oligodendrocytes, however considering the lack of presence of neutral lipids from astrocyte in other surrounding cells, it is thought astrocytes do not need to produce such lipids under physiological conditions. In addition, it was found that the medial, and to a lesser extent the lateral, habenula was targeted by fatty acids and did not show discrimination in uptake of saturated or unsaturated fatty acids. This is unlike uptake from the hypothalamus, which is also in close contact with the ventricular system.

 

Authors are unclear as to the physiological ramifications of these findings. Profound lipid uptake in the habenula may have links to external stimuli processing, which is supported by the expression of cannabinoid receptors in medial habenula, however the authors can only speculate this conjecture. More notably, the authors have designed a robust system of lipid based labeling that can simulate real lipid movements in cerebral tissues. This tracing system allows lipid transport to be more observable than previously available, and provide groundwork for future work.

Access the article here:

https://www.nature.com/articles/s41598-017-11103-5

 

References:

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