Flux of cholesterol, oxysterols and plant sterols across the blood-brain barrier and metabolic consequences

Brain is the cholesterol-richest organ and contains one fourth of the total body cholesterol. The majority of brain cholesterol is present in myelin that forms myelin sheathes around neuronal axons. This large pool of cholesterol present in brain is separated from body cholesterol by the blood brain barrier (BBB). This barrier prevents cholesterol, among many other substances, to flux from the circulation to the brain. BBB is composed of specific cellular and molecular components that allows it to perform its function.

Oxysterols are oxygenated cholesterol derivatives that play significant regulatory roles. 24S-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC) are two important oxysterols. Unlike cholesterol they have the ability to cross BBB. 24-OHC is produced exclusively in brain and it fluxes out to the circulation. 27-OHC is produced by all tissues including neural tissues but there is a net flux from circulation to the brain. These two oxysterols have been implicated to be important in some neurodegenerative diseases such as Alzheimer's disease where 24-OHC is thought to be protective and 27-OHC is blamed for some negative effects.

27-OHC is effeciently metabolized in the brain by a number of enzymes to produce 7α-hydroxy-3- oxo-4-cholestenoic acid (7-Hoca). This steroid acid has also the ability to cross the BBB to flux back to the circulation. Part of 7-Hoca fluxes to CSF where it represents the most abundant cholesterol metabolite. High levels of this compound were found in chronic subdural hematoma (CSH) samples.

Plant sterols are structurally similar to cholesterol. They are synthesized by plant cells only while animals obtain them from diet. Plant sterols are able to cross the BBB from blood to settle in the brain.

Disturbances in cholesterol metabolism, both cerebral and extracerebral, have been linked to neurodegenerative diseases such as Alzheimer's disease, Huntington’s disease and Parkinson’s diseases. BBB may also be affected in such diseases. Consequences of BBB disruption on cholesterol metabolism have never been studied previously.

In Paper I and Paper II, the levels of 7-Hoca in different neurological diseases were measured with a newly developed assay based on isotope dilution mass spectrometry. The level of this compound in patients with Alzheimer’s disease and vascular dementia were similar to controls. 7-Hoca was, however, elevated in a group of patients with different conditions involving BBB dysfunction. The possibility is discussed that 7-Hoca in CSF can be used as a diagnostic marker for conditions with a dysfunctional BBB.

Two alternative mechanisms for the elevated levels of 7-Hoca are suggested. There may be a primary increase in the flux of 27-OHC across the disrupted barrier followed by increased formation of 7- Hoca. The other possibility is a primary increase in the flux of albumin across the disrupted BBB followed by binding of 7-Hoca from the brain to the albumin.

Investigations were made to elucidate the mechanism behind of 7-Hoca accumulation in CSH. A very efficient binding of 7-Hoca to albumin could be demonstrated in two model experiments in-vitro. A high ratio between 7-Hoca and albumin could be demonstrated in the CSH from patients. The results suggest that the high affinity between 7-Hoca and albumin is the driving force for the accumulation of 7-Hoca in CSH.

In Paper III and Paper VI, characterization of a mouse model with BBB deficiency was performed. This mouse model (Pdgfbret/ret) lacks pericyte which is an essential component of BBB and therefore it ends up with a leaking BBB. Diet treatments were applied and brain, liver, plasma and other organs were inspected. Results show that plasma cholesterol reaches brain parenchyma in those animals while more 24-OHC fluxes through the defective BBB to the circulation. This effect leads to increased cholesterol synthesis in the brain. A theoretical model for regulation of cholesterol synthesis in a brain with BBB disruption is suggested. Plant sterols levels were found to be significantly increased in the brain of BBB deficient mice. Flux of two plant sterols across the BBB was studied in more detail. Pdgfbret/ret and control mice were fed diet mixed with deuterium labeled campesterol and sitosterol. Results show a time-dependent accumulation of these sterols in brain. More plant sterols were shown to pass across the defective barrier than across the normal one. Campesterol was found to cross both normal and disrupted barrier more effectively than sitosterol.

To summarize the present thesis illustrates the capacity of some specific cholesterol metabolites and plant sterols to pass the BBB and in the former case also membranes surrounding subdural hematomas. It also demonstrates presence of a cross-talk between the isolated pool of cholesterol in the brain and extracerebral pools of cholesterol. Furthermore it emphasizes the role of albumin binding for the flux of a steroid acid (7-Hoca) in the brain. The investigations also support the contention that 24-OHC is of importance for the regulation of cholesterol synthesis in the brain.

List of scientific papers

I. SAEED, A., FLORIS, F., ANDERSSON, U., PIKULEVA, I., LOVGREN- SANDBLOM, A., BJERKE, M., PAUCAR, M., WALLIN, A., SVENNINGSSON, P. & BJORKHEM, I. 2014. 7alpha-hydroxy-3-oxo-4- cholestenoic acid in cerebrospinal fluid reflects the integrity of the blood- brain barrier. J Lipid Res, 55, 313-8.
https://doi.org/10.1194/jlr.P044982

II. SAEED, A. A., EDSTRÖM, E., PIKULEVA, I., EGGERTSEN, G. & BJORKHEM, I. 2016. On the importance of albumin binding for the flux of 7 alpha hydroxy-3-oxo-4-cholestenoic acid in the brain. [Manuscript]

III. SAEED,A.A.,GENOVE,G.,LI,T.,LUTJOHANN,D.,OLIN,M.,MAST, N., PIKULEVA, I. A., CRICK, P., WANG, Y., GRIFFITHS, W., BETSHOLTZ, C. & BJORKHEM, I. 2014. Effects of a disrupted blood- brain barrier on cholesterol homeostasis in the brain. J Biol Chem, 289, 23712-22.
https://doi.org/10.1074/jbc.M114.556159

IV. SAEED, A. A., GENOVE, G., LI, T., HULSHORST, F., BETSHOLTZ, C., BJORKHEM, I. & LUTJOHANN, D. 2015. Increased flux of the plant sterols campesterol and sitosterol across a disrupted blood brain barrier. Steroids, 99, 183-8.
https://doi.org/10.1016/j.steroids.2015.02.005