Month: February 2019

Lipids on the Mind

Lipids on the Mind

Blog Spotlight #1

Access the article: https://www.nature.com/articles/s41598-018-26636-6.pdf?origin=ppub


Image obtained from: Lacombe, R. J. Scott, Raphaël Chouinard-Watkins, and Richard P. Bazinet. “Brain Docosahexaenoic Acid Uptake and Metabolism.” Molecular Aspects of Medicine, Dietary fatty acids, lipid mediators, cell function and human health, 64 (December 1, 2018): 109–34. https://doi.org/10.1016/j.mam.2017.12.004.

               In the United States alone, more than 200,000 people are diagnosed with brain cancer every year, a common metastasis site for patients with advanced primary lung cancer, breast cancer, or melanoma. Many of these metastases result from other primary sources and approximately 50% of lung and melanoma patients and 20% of breast cancer patients develop secondary lesions in the brain. A common feature in most brain metastases is resistance to therapy, which can be attributed to the poor penetration of therapeutics across the blood-brain barrier (BBB). This research is most impactful since there is very little understood about the mechanisms that regulate BBB permeability in normal brain tissue or brain malignancies. This prevents exploitation of the BBB for drug delivery, a method that could be useful for the treatment of many neurodegenerative or malignant brain diseases. Animal models make tumor pathophysiology comparison difficult, relying on both mouse and human cell lines for research purposes.

               Major facilitator superfamily domain‐containing protein‐2a (Mfsd2a) has recently gained the spotlight for its regulatory role in the maintenance of proper functioning of the BBB (Ocak et al., n.d.). Remarkably, Mfsd2a has been implicated in the mediation of the blood–brain barrier (BBB) permeability by selective transportation of the lysophosphatidylcholine (LPC) fatty acids (Nguyen, et al., 2014). The gene, MFSD2a plays an important role in mammalian tissue and organ growth, lipid metabolism and cognitive and motor functions. Specifically, in the brain and retina, Mfsd2a selectively transports the omega-3 fatty acid docosahexaenoic acid (DHA) across the BBB. This was confirmed by experimenting with a genetic deletion of Mfsd2a mice which led to impaired DHA transport and reduced levels of certain lipid metabolites. Loss-of-function studies provide concrete evidence in support of pursuing this pathway.

               Loss-of-function (including familial mutations) in human MFSD2A are linked to cognitive deficits and ataxia due to deficiencies in DHA transport and metabolism. As we learned in BCM441, DHA cannot be synthesized in the brain, thus it must be transported there by other means. DHA accumulated in the brain is selectively derived from the plasma where it is bound to albumin as an unesterified fatty acid or to LPC. DHA is a crucial polyunsaturated omega‐3 fatty acid required for brain development, motor, and cognitive functioning. A previous study reported down-regulation of Mfsd2a in a pericyte-deficient mouse model, suggesting that pericytes are necessary for the induction or maintaining Mfsd2a gene expression. For this reason, the authors set out to investigate the association between pericytes and endothelial cells within the vasculature of metastatic brain tumors.

Figure 1. The regulation of BBB permeability is mediated by Mfsd2a suppression of caveolae‐mediated transcytosis in endothelial cells. Essential DHA is absorbed from the intestines or synthesized, conjugated to LPC in the liver and bound to albumin forming LPC‐DHA in the plasma. The significant pathway of LPC‐DHA uptake across the endothelial cells of the BBB is controlled by Mfsd2a. The lipid composition of the plasma membrane which is highly enriched by DHA impairs the formation of caveolae vesicles. In the absence of Mfsd2a, transcytosis across the endothelial cell increases. BBB: blood-brain barrier, DHA: Docosahexaenoic acid, LPC: Lysophosphatidylcholine.

               The researchers analyzed brain metastases using patient-derived xenograft (PDX) which is a tissue graft from a donor of a different species from the recipient. In this case mouse models were used to study signaling pathways involved in disruption of the intratumoral BBB. Cells were initially grown in serum-free media, with the intention of relieving the effects of substances such as growth factors on the cells. Cultured metastatic tumor cells grew as neurosphere-like spheroids in serum-free media and expressed epithelial markers including E-Cadherin and β-catenin. Specific patterns of localization and expression of Mfsd2a in brain endothelial cells suggested a functional role that was further investigated. Since intratumoral blood vessels showed abnormal BBB properties, the authors analyzed the expression of Mfsd2a in metastatic tumors and compared these results to the opposite, non-injected hemisphere. The results evidenced a statistically significant decrease of Mfsd2a protein expression in vascular endothelial cells from brain metastases originating from primary breast cancer.

               Importantly, the results containing reduced numbers of perivascular astrocytes strongly correlated with lack of Mfsd2a expression in brain metastasis endothelial cells. The data supports the notion that endothelial cells of brain metastases are in contact or are associated with pericytes but show diminished interactions with perivascular astrocytes. As determined previously, the presence of pericytes was confirmed to be required for the expression of Mfsd2a in the brain endothelial cells. The analysis of previous microarray data of pericyte‐deficient mouse models (Armulik et al., 2010) demonstrated a significant decrease in Mfsd2a expression in pericyte‐deficient animals (Ben-Zvi et al., 2014). This data can be corroborated with reduced expression of Mfsd2a. Further, astrocytes have been known to secrete cytokines and growth factors that modulate BBB properties in the brain vascular endothelium. For this reason, the researchers examined the influence of conditioned media taken from primary mouse brain astrocytes or human astrocytes on the expression of Mfsd2a in low passage. This means that the cells were not passed or split more than five times in order to keep mutations and selective pressures at a minimum to control for mutations. This demonstrated that both mouse and human astrocytes secrete factors that promote the expression of Mfsd2a mRNA in HBMECs.

               It is also known that Mfsd2a is a transporter for DHA when it is conjugated to lysophosphatidylcholine (LPC) in circulation, as described by the pathway in Figure 2. Thus, the effects of astrocyte conditioned media on Mfsd2a-dependent uptake of nitrobenzoxadiazole-LPC (NBD-LPC) were examined using a fluorescent tracer, revealing an increased uptake of NBD-LPC by HBMECs treated with astrocyte-conditioned media compared to a control non-conditioned media. The astroglial-derived factors that positively regulate the expression of Mfsd2a were determined by treating HBMECs with different cytokines with known effects on endothelial barrier properties, such as VEGF, bFGF/FGF2, and TGFβ1. After treatment, levels of Mfsd2a gene expression were measured using qRT-PCR. It was concluded that both bFGF and TGFβ1 have the ability to induce the expression of MFSD2A in HBMECs, whereas VEGF had an inhibitory effect on MFSD2A gene expression.

Figure 2. The proposed mechanism for Mfsd2a‐mediated LPC transport across the plasma membrane is Na dependent. Na binds to Mfsd2a and LPC inserts itself into the outer membrane and diffuses laterally into the hydrophobic cleft of Mfsd2a.
this causes a conformational change in Mfsd2a to an inward‐open form while the zwitterionic headgroup of LPC is inverted within the transporter (Quek et al., 2016). Na: sodium.

               Finally, to evaluate a direct effect of DHA on metastases, the authors treated cultured primary lung and breast brain spheroids with DHA. They observed decreased growth and survival of tumor cells treated with DHA compared to controls. Additionally, DHA treatment positively impacted the growth and survival of HBMECs. Therefore, the researchers conclude that loss of Mfsd2a in metastatic tumor endothelial cells leads to decreased uptake of essential fatty acids, specifically DHA, which promotes tumor growth and survival in the brain microenvironment.

               Mediating the transport of DHA is not the only function, however. In addition, Mfsd2a suppresses caveolin-dependent transcytosis. Increased vesicles in Mfsd2a‐deficient mice have been found to be positive for caveolin‐1 (cav‐1) which is the mandatory protein coat implicated in the formation of caveolae vesicles from the plasma membrane (Ocak et al., n.d.). DHA was also previously shown to displace cav‐1 from the plasma membrane, decreasing the formation of caveolae vesicles in its presence. This was demonstrated using a genetic deletion of murine Mfsd2a leading to enhanced transcellular transport and breakdown of the vascular endothelial barrier in the brain and retina. The research shows that Mfsd2a expression as well as its transport functions are down-regulated in the metastatic brain tumor vascular endothelium and can be explained by the absence of astrocytes that use TGFβ1 and bFGF signaling to maintain expression of Mfsd2a in cerebral endothelial cells.

               Lipid transport pathways of Mfsd2a pay a critical role in the regulation of permeability and in the maintenance of the integrity of the BBB by acting as a suppressor on caveolae‐mediated transcytosis in the endothelial cells of the CNS vasculature. Therefore, loss of MFSD2A promotes metastatic tumor growth and survival in the brain microenvironment by altering DHA transport and metabolism. Mfsd2a is a novel LPC transporter selectively expressed in the endothelial cells of the CNS and provides impactful contributions to the formation, functioning, and maintenance of the BBB. Looking ahead to the future, this pathway can possibly be harnessed for pharmaceutical delivery across the BBB. Changes in Mfsd2a expression levels following different types of brain injury may be unique to the pathology, which will be an important factor to consider while creating specific targeted therapeutic strategies. Nonetheless, large numbers of in vivo and in vitro studies are warranted in order to corroborate the practicality and applicability of pharmacologic strategies based on the modulation of BBB permeability via Mfsd2a. Restoring DHA and or its metabolites to the tumor microenvironment may serve as a treatment option for patients with metastatic brain cancer.

References:

1. Ocak, P.E., Ocak, U., Sherchan, P., Zhang, J.H., Tang, J., n.d. Insights into major facilitator superfamily domain-containing protein-2a (Mfsd2a) in physiology and pathophysiology. What do we know so far? J. Neurosci. Res. 0. https://doi.org/10.1002/jnr.24327

2. Pericytes regulate the blood–brain barrier | Nature [WWW Document], n.d. URL https://www.nature.com/articles/nature09522 (accessed 2.11.19).

3. Structure and function of the blood–brain barrier – ScienceDirect [WWW Document], n.d. URL https://www.sciencedirect.com/science/article/pii/S0969996109002083?via%3Dihub (accessed 2.11.19).

4. Tiwary, S., Morales, J. E., Kwiatkowski, S. C., Lang, F. F., Rao, G., & McCarty, J. H. (2018). Metastatic brain tumors disrupt the blood‐brain barrier and alter lipid metabolism by inhibiting expression of the endothelial cell fatty acid transporter Mfsd2a. Scientific Reports, 8(1), 8267. https://doi.org/10.1038/s41598-018-26636-6

5. Nguyen, L. N., Ma, D., Shui, G., Wong, P., Cazenave‐Gassiot, A., Zhang, X., … Silver, D. L. (2014). Mfsd2a is a transporter for the essential omega‐3 fatty acid docosahexaenoic acid. Nature, 509(7501), 503–506. https://doi.org/10.1038/nature13241