Endogenous murine Lcat mRNA is found in brain and in primary astrocytes and neurons. A: Total Lcat mRNA levels in various tissues were determined by quantitative RT-PCR (qRT-PCR) and normalized to actin mRNA. Graph summarizes the results of four independent experiments with a total of N = 12 samples for brain and liver, and N = 4 for spleen, lung, kidney and intestine, all measured in duplicate. Lcat mRNA expression levels are expressed as fold difference over brain. B: Total mRNA of primary neurons, astrocytes, and microglia was purified using Trizol and tested for the presence and levels of murine Lcat mRNA by qRT-PCR. Individual samples were normalized to actin mRNA to correct for cell number variability. Graph summarizes the results of two independent experiments with a total of N = 10 individual cultures for neurons, N = 7 for astrocytes, and N = 8 for microglia, all measured in duplicate. Lcat mRNA expression levels are expressed as fold difference over astrocytes. Error bars represent SEM. One-way ANOVA with a Tukey posttest was used for statistical analysis. *** P < 0.0001. C: Fifteen microliters of 100-fold concentrated conditioned media of LCAT-deficient glia (LCAT^−/−, negative control) (N = 1), wild-type (WT) astrocytes (N = 2), and microglia (N = 2), and 1.5 microliters of neuronal-conditioned media (N = 2) were evaluated for LCAT protein levels by Western blot. The arrow demarcates LCAT protein, and the 55 kDa species detected in several lanes is a nonspecific cross-reactive band.

Apolipoprotein E (apoE) is the main LCAT activator in glia-conditioned media (GCM). A: Cholesterol esterification was evaluated in conditioned media from WT, apoE-deficient (apoE^−/−) and LCAT-deficient (LCAT^−/−) mixed glial cultures. The first two bars on the graph represent the level of esterification seen in conditioned media supplemented only with [3]Hcholesterol-labeled EYPC/UC vesicles, whereas the last three bars correspond to the effects seen when [3]Hcholesterol-labeled EYPC/UC vesicles were complexed with 5 μg of human apolipoprotein A-I (apoA-I) purified from plasma and then added to the conditioned media. Graph represents at least four independent experiments with an N ⩾ 4 independent cultures for each group. B: Cholesterol esterification was evaluated in conditioned media from WT and apoE-deficient (apoE^−/−) mixed glial cultures after addition of [3]Hcholesterol-labeled EYPC/UC vesicles and increasing amounts of purified human apoA-I (added only to apoE-deficient samples). Graph represents the MER of two independent experiments. One-way ANOVA with a Dunnett posttest was used for statistical analysis. ** P < 0.05 compared with first WT group (A) and WT (B).

LCAT-deficiency leads to altered lipoprotein metabolism in GCM and cerebrospinal fluid (CSF). A: Distribution of apoE-containing particles in WT and LCAT-deficient (LCAT^−/−) mixed glial cultures was evaluated by native gel electrophoresis. WT(−) and LCAT^−/−(−) lanes represent WT and LCAT-deficient media with no treatment. Incubation of LCAT^−/− media with exogenous LCAT (BHK-LCAT media) at 37C for 1 h results in the disappearance of an 8 nm particle observed in LCAT^−/− media. This effect is not seen when LCAT^−/− media is incubated with vehicle alone (control BHK media), suggesting that this particle may represent the preferred LCAT substrate. B: Distribution of apoE and apoA-I-containing particles in the CSF of WT and LCAT^−/− animals was evaluated by native gel electrophoresis. Blots are representative of two independent experiments and at least four animals per genotype. C: Total cholesterol levels were evaluated in CSF from WT, LCAT^−/−, and ABCA1^−/− mice by mass spectrometry. Data represent two independent experiments with N ⩾ 6 animals per group. * P < 0.05 with respect to WT, as determined by one-way ANOVA with a Dunnett posttest analysis.

Primary astrocytes are the main source of active LCAT in vitro. A: Astrocyte conditioned media (ACM), neuronal conditioned media (NCM), and microglia conditioned media were evaluated for the ability to esterify cholesterol on [3]Hcholesterol-labeled egg yolk phosphatidylcho1ine (EYPC)/unesterified cholesterol (UC) vesicles. Graph summarizes two independent experiments with N = 5 individual cultures per cell type. Cholesterol esterification is expressed as molar esterification rate of cholesterol (MER) (nmoles of cholesterol esterified per h per ml of concentrated media) per microgram for cellular protein, to correct for differences in cell number between cultures. B: Cholesterol esterification detected in ACM is LCAT dependent. ACM from WT and LCAT-deficient (LCAT^−/−) glia cultures were evaluated for the ability to esterify cholesterol as above. Graph summarizes the results of four independent experiments. Cholesterol esterification is expressed as MER. One-way ANOVA with a Tukey posttest was used for statistical analysis. *** P < 0.0001.

ApoE and ABCA1 are required for the generation of LCAT substrates by glia cells. A: Time-dependent esterification of cholesterol on nascent lipoproteins from WT mixed glia in the presence of recombinant LCAT derived from baby hamster kidney cells stably transfected with human LCAT (BHK-LCAT cells). No esterification activity is observed in the absence of nascent GCM lipoproteins in either BHK or BHK-LCAT control media. Graph represents two independent experiments and expresses % of cholesterol esters (CE). B: Cholesterol esterification by recombinant LCAT on glia-derived lipoproteins was evaluated as above over a period of 5 h in WT, apoE-deficient (apoE^−/−), and ABCA1-deficient (ABCA1^−/−) glia conditioned media. Graph represents two independent experiments. C: In situ cholesterol esterification in mixed glial conditioned media. WT, LCAT^−/−, and apoE^−/− glial cultures were labeled with [3H]cholesterol for 18 h prior to concentration and separation of UC and CE by TLC. Graph represents two independent experiments. *** P < 0.0001 with respect to WT, as determined by one-way ANOVA with a Dunnett posttest analysis.