ROLE OF LECITHIN:CHOLESTEROL ACYLTRANSFERASE (LCAT) IN BRAIN CHOLESTEROL METABOLISM AND ITS INVOLVEMENT IN ALZHEIMER¿S DISEASE

Cholesterol is an essential component of both peripheral and central nervous system (CNS). Brain cholesterol is synthesized in situ and is almost completely isolated from plasma, but a little fraction can exchange through the blood-brain barrier. The role of esterification in cholesterol metabolism and turnover in the brain remains unknown. Lecithin:cholesterol acyltransferase (LCAT) is the unique enzyme responsible for the esterification of sterols in biological fluids and plays a key role in cholesterol metabolism. In the brain apoE is the main LCAT cofactor, and apoE4, one of the three apoE isoforms, is the strongest known genetic risk factor for Alzheimer’s disease (AD), but its ability in activation of LCAT enzyme has never been investigated. Moreover, several epidemiological studies indicate a strong inverse association between the risk of developing AD and plasma HDL-c levels. The mechanism by which plasma HDL influence the pathogenesis and progression of AD remains unsolved and the crucial step of cholesterol esterification exerted by LCAT in HDL metabolism could be involved. The purpose of this thesis was to investigate the role of LCAT in CNS with a specific focus on its activation by different apoE isoforms, and to evaluate cholesterol esterification, cholesteryl esters (CEs) composition in plasma and cerebrospinal fluid (CSF) in AD. To highlight the contribution of different apoE isoforms in the activation of LCAT, plasma cholesterol esterification rate (CER) was evaluated in 311 controls who express functional LCAT and either apoE2, apoE3, or apoE4 isoforms. In addition, the kinetic of LCAT activity towards reconstituted HDLs (rHDLs) containing each apoE isoform was determined using the Michaelis-Menten model. In order to clarify the involvement of LCAT in AD, 70 AD patients and 74 cognitively normal controls comparable for age and sex were enrolled. Lipids and lipoprotein profile, cholesterol esterification, and cholesterol efflux capacity (CEC) were evaluated in plasma and CSF using assays set for measurement in plasma, which were appropriately modified for CSF. CEs and phospholipids (PLs) species were evaluated with omics techniques, LC-MS and LC-MS/MS respectively, in plasma and CSF of a subgroup of AD and controls. Plasma CER of controls was ~20% higher in apoE2 carriers compared to apoE3 carriers, and ~30% higher in apoE2 carriers compared to apoE4 carriers. After adjusting for age, sex, total cholesterol, HDL-c, apoA-I, apoB, chronic kidney disease diagnosis, zygosity, and LCAT concentration, CER remained significantly different among carriers of the three apoE isoforms. rHDLs containing apoE2 were associated with a lower affinity but higher maximal esterification rate, compared to particles containing apoE3 or apoE4. AD patients have normal plasma lipids, but significantly reduced unesterified cholesterol (UC) and unesterified/total cholesterol ratio (UC/TC). LCAT activity and CER, were reduced by 29% and 16%, respectively, in plasma of AD patients. Plasma HDL subclass distribution in AD patients was comparable to that of controls, but the content of small discoidal preβ-HDL particles was significantly reduced. In agreement with the reduced preβ-HDL particles, CEC mediated by the transporters ABCA1 and ABCG1 was reduced in AD patients’ plasma. The CSF UC/TC was increased in AD patients, and CSF CER and CEC from astrocytes were significantly reduced in AD patients. In the AD group, a significant positive correlation was observed between plasma UC and UC/TC ratio with Aβ1-42 CSF content. Cholesteryl esters composition in CSF of AD revealed significant enrichment in saturated fatty acids (FA) and monounsaturated FA and a depletion in polyunsaturated FA in cholesterol species compared to controls. Specifically, cholesteryl ester palmitate and oleate were increased (p=0.001; p=0.005); while linoleate, the major LCAT substrate, was reduced (p=0.029) in AD. Plasma samples did not show any difference in CEs composition and CSF PLs species analysis displayed no significant modification. The present results suggest that the apoE2 isoform is associated with a higher LCAT-mediated cholesterol esterification. This observation may contribute to the characterization of the peculiar functional properties of apoE2, which has been demonstrated to be protective against AD. The data of AD patients indicate that cholesterol esterification is hampered in plasma and CSF and that plasma cholesterol esterification biomarkers (UC and UC/TC ratio) are significantly associated with disease biomarkers (i.e., CSF Aβ1-42). CEs composition confirmed the crucial role of cholesterol esterification in AD, providing new insights and possibilities for cholesterol esterification as a new pharmacological target in AD.