Plasma and cerebrospinal fluid cholesterol esterification is hampered in Alzheimer’s disease

Brain cholesterol metabolism is segregated from the peripheral circulation by the blood–brain barrier (BBB) and cholesterol transport in the central nervous system (CNS) is operated by lipoproteins very similar to the circulating high-density lipoproteins (HDLs) and called “HDL-like particles” [1]. Plasma HDLs have been extensively studied and characterized and consist of heterogeneous particles differing in shape, size, and composition [2]. The heterogeneity of circulating HDLs is the result of HDL remodeling by various enzymes and lipid transfer proteins. Among these factors, lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible of cholesterol esterification in plasma, plays a major role in HDL maturation [3].

Brain HDLs are similarly heterogeneous in size and composition [1]; while in plasma the major HDL protein constituent is apolipoprotein A-I (apoA-I), the major protein component of brain HDL is apoE [1], which is synthesized in situ and does not derive from the circulation [1]. In humans, apoE exists as three different isoforms, apoE2, apoE3, and apoE4, which differ by amino acid substitutions at positions 112 and 158 in the mature protein [4]. ApoE3 contains cysteine at position 112 and arginine at position 158, whereas apoE2 and apoE4 contain cysteine or arginine, respectively, at both positions. ApoE3 is the most common isoform, whereas the apoE2 and apoE4 isoforms are less frequent and have altered functionality. ApoE4 has reduced lipoprotein binding and it has been associated with hypercholesterolemia and Alzheimer’s disease (AD) [1, 5‐9]. Brain HDLs also contain apoA-I, which is not produced in the CNS but which, in the poorly lipidated form, can cross the BBB, likely through different mechanisms [10]. ApoJ, known as clusterin, is also synthesized in the brain and it is the second major apolipoprotein constituent of brain lipoproteins [11]. Brain HDL remodeling likely occurs by the action of the same enzymes and proteins operating in the circulation. LCAT, the major player in HDL maturation, is mainly synthesized by the liver but also in the brain, suggesting a crucial role of this enzyme in the maturation of brain lipoproteins, although LCAT concentration in the cerebrospinal fluid (CSF) is much lower than in plasma [12].

An inverse association between plasma HDL-cholesterol levels and the risk of dementia and AD has been reported, although with conflicting results [1], but an involvement of circulating HDLs in AD etiology and progression has never been proven. Few studies have analyzed HDL in the CSF, for obvious reasons of sample accessibility. Published data have mainly focused on the cholesterol efflux capacity (CEC) of CSF HDL and showed that it is reduced in AD patients [13, 14]; a very recent report has suggested that the CSF levels of apoJ are a determinant of the low CSF CEC observed in dementia [15].

In the present report, we have hypothesized that cholesterol esterification is altered in AD plasma and CSF. Due to the much lower concentration of lipids and enzymes in the CSF compared to plasma, we have set appropriate techniques to measure lipids, lipoproteins, apolipoproteins, and cholesterol esterification in the CSF.

Brain contains a huge amount of cholesterol, 70% of which is present in myelin. The remaining 30% of brain cholesterol is distributed between glial cells and neurons, which in the mature state lose their ability to produce cholesterol and must rely for cholesterol supply on glial cells, mainly astrocytes [31]. Cholesterol transport in the brain is handled by lipoproteins very similar to circulating HDLs, mainly synthesized in situ but also deriving from the periphery [1]. Brain lipoproteins undergo a remodeling process like the one occurring in plasma, mediated by enzymes and lipid transfer proteins which are also expressed in the brain [1].

Cholesterol esterification is a fundamental step in cholesterol transport in plasma and indeed 70% of circulating cholesterol is esterified. Cholesterol esterification in humans is operated by three distinct enzymes: LCAT, which acts in plasma and other biological fluids, and sterol O-1 and sterol O-2 acyltransferases (SOAT1 and SOAT2), which are intracellular enzymes. LCAT is expressed mainly in the liver and is responsible for the formation of most of the plasma CEs, as shown by the almost absence of CEs in genetic LCAT deficiency [32]. SOAT1 is expressed in all cell types, while SOAT2 is expressed in hepatocytes and enterocytes, and in humans it has little contribution to circulating CEs [33]. Dysregulation of cholesterol metabolism in the brain has been associated with AD and other neurodegenerative disorders [1], but the mechanism(s) linking cholesterol metabolism and disease pathogenesis are not well understood.

Here we show for the first time that cholesterol esterification is altered in plasma and CSF of AD patients, and that these alterations strongly correlate with CSF AD biomarkers. LCAT is the enzyme responsible of almost all circulating CEs, thus abnormalities in plasma cholesterol esterification parameters (i.e., unesterified cholesterol levels and unesterified/total cholesterol ratio) likely derive from dysregulation of the LCAT system. Indeed, we have found reduced LCAT activity in the plasma of AD patients, which could be explained by a selective reduction of plasma unesterified cholesterol, the LCAT substrate, in the presence of normal concentrations of total cholesterol. Unesterified cholesterol is carried by all plasma lipoproteins, but mostly by small discoidal HDL particles, which are selectively reduced in AD patients’ plasma. Unesterified cholesterol in discoidal HDLs primarily derives from cell cholesterol efflux via the active ABCA1- and ABCG1-mediated pathways [34]. In the present cohort of AD patients, we have observed a reduced capacity of patients’ HDLs to promote transporter-mediated cell cholesterol efflux through these two pathways. The cholesterol esterification process in the brain is not well known, and the very low concentration of LCAT in the CSF makes it difficult to measure the process ex vivo. Using assays set for measurement in plasma appropriately modified, we first measured the CSF unesterified/total cholesterol ratio and the cholesterol esterification rate in CSF of cognitively normal controls and showed that CSF cholesterol esterification rate is much lower than in plasma, leading to a higher unesterified/total cholesterol ratio. The reduced CE formation in the CSF could be explained by the low LCAT concentration, and/or by the different substrate and activator. Indeed, small discoidal HDLs, the best LCAT substrate, are absent in the CSF, as shown by our HDL subclass analysis, and apoE, the most abundant apolipoprotein and likely LCAT activator in the CSF, is much less efficient than apoA-I in activating LCAT [35]. We next measured cholesterol esterification in the CSF of AD patients and found that CER was strongly reduced compared to controls, and the unesterified/total cholesterol was strongly increased in the CSF of AD patients, where around 50% of cholesterol is not esterified. As in the plasma, most of the CEs in the CSF should be LCAT-derived, thus suggesting a dysregulation of the LCAT system in AD CSF, which could be partly explained by the presence of unfunctional apoE in CSF of AD patients [36]. The lower content of CEs in AD CSF could also derive from the accumulation of CEs in the brain, and indeed excess CEs have been shown in brain vulnerable regions of AD patients [37]. The dysregulated esterification process may consequently halt the correct remodeling of HDL particles, with a negative impact on their cholesterol transport activity in the brain. In line with this hypothesis, we found that these particles were less efficient in promoting astrocyte cholesterol efflux, the first step of the HDL-promoted cholesterol transport towards neurons [27].

All together our results point to novel lipid-related factors implicated in AD, to be potentially used in the future as CSF or plasma biomarkers, in addition or as an alternative to those previously considered. Among these, total cholesterol and levels of HDL apolipoproteins, i.e., apoE, apo A-I, and apo J, have been already investigated [38, 39], leading to contradictory results on their association with AD [15, 40‐43]. Similarly, oxysterols, in particular the neuronal-derived 24S-hydroxycholesterol, have been proposed as markers of neurodegeneration in AD, although with conflicting results [44‐46]. Overall, these inconsistencies highlight the importance of identifying more reliable and powerful lipid biomarkers, that focus on peculiar and novel aspects of cholesterol metabolism, such as esterification.

In conclusion, we found that cholesterol esterification is hampered in plasma and CSF of AD patients and that plasma cholesterol esterification biomarkers (unesterified cholesterol and unesterified/total cholesterol ratio) are significantly associated to disease biomarkers (i.e., CSF Aβ_1-42). Such impairment is associated to defects in HDL functions, namely plasma and CSF cholesterol efflux capacity. Whether the dysregulation of cholesterol esterification is limited to AD or is a common trait in other neurodegenerative disorders remains to be defined. Taken together, the reported results suggest that LCAT-mediated cholesterol esterification could represent a potential target of novel therapeutic interventions in AD, although the causality between esterification abnormalities and related cognitive decline cannot be established from our data. A final caveat should be made, since only about a third of plasma samples were obtained from cognitively intact controls who were characterized by normal CSF biomarkers. In fact, albeit asymptomatic, some control subjects could express AD biomarkers many years before cognitive complaints may appear, possibly partially biasing our results.