Brain cholesterol metabolism : A study of mouse and man

Sammanfattning: The brain is the most cholesterol rich organ in the body and contains about one quarter of the body's total cholesterol. How this cholesterol and its metabolism affects the complex brain under normal and pathological conditions has been the focus of intense research in recent years due to its possible association with the development or progression of neurodegeneration. The primary objective of this thesis was to increase our understanding about cholesterol homeostasis in the brain and its relevance for neurodegenerative disease. Oxysterols are cholesterol oxidation products which are found in the body wherever there is cholesterol. The major oxysterol in brain is 24S-hydroxycholesterol which is involved in the elimination of excess cholesterol from the brain. Another oxysterol which may also prove important to cholesterol homeostasis in the brain, is 27-hydroxycholesterol. This oxysterol is formed from cholesterol by way of the cytochrome P450 enzyme, CYP27A1. To establish a sterol profile of the brain, cholesterol, cholesterol precursors and oxysterols, were measured by isotope dilution mass spectrometry in different areas of the brain of patients with advanced Alzheimer's disease (AD) and healthy controls. In AD, the levels of the oxysterols 24Shydroxycholesterol and 27-hydroxycholesterol in the brain were altered in comparison to controls. 24S-Hydroxycholesterol was decreased in AD, possibly due to the loss of neurons. A surprising result was the increased levels of 27-hydroxycholesterol in the AD brain. As CYP27A1 expression was not found to be increased in the brain, this led to the conclusion that there could be a flux of 27hydroxycholesterol from the circulation into the brain. To investigate a possible uptake of the oxysterol, 27-hydroxycholesterol, into the brain and its possible metabolism there, blood samples were simultaneously taken from the internal jugular vein and the brachial artery of healthy volunteers. The level of 24S-hydroxycholesterol was found to be higher in the venous samples, confirming previous studies that there is a net flux of this oxysterol out of the brain. 27-Hydroxycholesterol had higher concentrations in the arterial samples indicating a net flux of this oxysterol from the circulation into the brain. Preliminary findings also indicated that 7alphahydroxy3-oxo-4-cholestenoic acid may be fluxing out of the brain, a steroid which can be formed in the brain from 27hydroxycholesterol. Cholesterol 24-hydroxylase or CYP46A1, oxidises cholesterol to 24S-hydroxycholesterol, which unlike cholesterol, can freely traverse the blood brain barrier. How CYP46A I is regulated has not been clarified. A structural and functional characterisation of the promoter of the human CYP46A1 gene was carried out, and using a luciferase reporter assay a broad spectrum of regulatory axes, including cholesterol were investigated to determine if they could regulate the transcription of the gene. Under most conditions there was a failure to demonstrate significant transcriptional regulation. The inability of cholesterol to regulate CYP46A1 was supported in a mouse model with very low cholesterol levels (Dhcr24-/- mouse), which had a normal level of Cyp46a1 mRNA in the brain. In summary, cholesterol homeostasis in AD is disturbed as evident by the altered levels of oxysterols in the brain. While there is flux of 24S-hydroxycholesterol out of the human brain we have demonstrated here that there is also a corresponding flux of 27-hydroxycholesterol from the circulation into the brain and that it may be metabolised and eliminated again most probably in part as 7alpha-hydroxy-3-oxo-4-cholestenoic acid. Finally CYP46A1 is important in cholesterol elimination from the brain but cholesterol itself fails to regulate it.