LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice.

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT(-/-) mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer's-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.

Plasma ␣ -HDL and apoA-I levels are dramatically reduced in LCAT Ϫ / Ϫ mice ( 44,45 ), however the macrophage reverse cholesterol transport pathway is largely preserved ( 46 ). Whether LCAT is pro-or antiatherogenic remains contested, as results from both animal and human studies are confl icting ( 43 ).
In the CNS, we have previously shown that LCAT is secreted by astrocytes and is capable of esterifying FC contained on glial-derived nascent apoE-containing particles ( 47 ). apoE is hypothesized to be the major LCAT activator in the CNS, as apoE is suffi cient to stimulate esterifi cation of endogenous cholesterol in glial-conditioned media ( 39 ). apoA-I, which is not synthesized within the CNS but is found in CSF, is also capable of esterifying cholesterol using glial-derived LCAT ( 47 ). The levels and activity of LCAT in CSF are estimated to be 2.2-2.5% of serum LCAT ( 42,48 ). In young C57Bl.6 mice, LCAT defi ciency leads to a dramatic increase in apoE and a concurrent decrease of apoA-I levels in CSF ( 47 ), suggesting that LCAT activity may in part regulate CSF apoE and apoA-I levels.
LCAT activity in the CSF of AD subjects has been reported to be up to 50% lower than in cognitively normal subjects ( 42 ). It is not known whether this association may be related to A ␤ metabolism, such that reduced LCAT may impair A ␤ clearance. It is also not known whether aging or the presence of A ␤ may impair LCAT activity. To address the question of whether A ␤ clearance requires LCATmediated maturation of apoE-bearing HDL-like particles, we assessed the impact of LCAT defi ciency on soluble and aggregated A ␤ levels as well as parenchymal and vascular amyloid burden in the APP/PS1 model of AD. We also assessed the impact of aging and A ␤ accumulation on LCAT expression and activity. Here we report that protein levels and plasma activity of LCAT are not altered by age and/or A ␤ accumulation and that neither A ␤ nor amyloid levels are increased in the absence of LCAT. We conclude that LCATmediated cholesterol esterifi cation and maturation of apoEcontaining lipoproteins in the CNS is not required for apoE's role in A ␤ metabolism.

Animals
APP/PS1 (line 85) mice (Jackson Laboratories), which coexpress two transgenes from the murine prion promoter: a chimeric mouse/human APP cDNA containing the Swedish (K670M/ N671L) mutations and the human PS1 gene deleted for exon 9, were crossed with LCAT Ϫ / Ϫ animals followed by one backcross to

CSF, plasma, and tissue collection
Mice were anesthetized by intraperitoneal administration of a mixture of 20 mg/kg xylazine (Bayer) and 150 mg/kg ketamine With respect to apoE levels, most studies suggest that apoE promotes retention of A ␤ in the brain, as brain-toblood transport of radiolabeled A ␤ injected into the brain is slowed when premixed with human apoE compared with free A ␤ ( 20,21 ). Also supporting this viewpoint is the observation that amyloid burden is lower in hemizygous APOE3 and APOE4 APP/PS1-21 and hAPP J20 AD mice compared with homozygous controls ( 17,22 ). Intriguingly, as a recent microdialysis study showed that very little apoE is actually associated with soluble A ␤ in brain interstitial fl uid, apoE may retard A ␤ clearance from brain to blood by competing with A ␤ for binding to the LDL receptorrelated protein 1 (LRP1) ( 23 ). However, another study found that there was signifi cant interaction between apoE and A ␤ , and that the levels of soluble apoE-A ␤ decreased in an isoform-specifi c manner apoE4<apoE3<apoE2 ( 24 ). These observations suggest that the putative interactions between apoE and A ␤ may vary depending on the experimental conditions and pools of apoE and A ␤ that are studied. Additionally, brain-specifi c overexpression of the LDL receptor (LDLR), the major apoE receptor ( 25 ), leads to signifi cantly decreased brain apoE levels and signifi cantly reduced amyloid and A ␤ loads, presumably by accelerating apoE uptake ( 26,27 ).
How apoE is involved in A ␤ metabolism is not completely understood, as net levels of apoE are only one part of the equation; the degree to which apoE is lipidated also significantly affects function. Lipidation of apoE in the CNS is performed by ABCA1 ( 28,29 ), akin to the well-established role of ABCA1-mediated lipidation of apoA-I in the periphery ( 30 ). Plasma and CSF apoA-I levels are markedly reduced in mice defi cient in ABCA1, whereas CSF and brain tissue apoE are decreased by 60-80% ( 28,29,31 ). ABCA1 Ϫ / Ϫ mice display increased insoluble A ␤ and amyloid deposition when crossed onto the TgSwDI ( 32 ), APP23 ( 33 ), and PDAPP ( 34 ) mouse models of AD. Conversely, selective overexpression of ABCA1 by 6-fold or more is suffi cient to prevent the formation of amyloid plaques without altering the net levels of CNS apoE or apoA-I in PDAPP mice ( 35 ).
Similar to apoA-I-containing HDL particles in plasma, apoE-containing HDL-like particles in the CNS exist in two major structural conformations depending on their maturation state ( 3 ). In vitro, astrocytes, and to a lesser extent, microglia, secrete several nascent discoidal apoE particles ranging from 7.5 to 17 nm in diameter that contain 0-18% of their cholesterol as esters (36)(37)(38)(39). By contrast, apoE-and apoA-I-containing lipoprotein particles in CSF are 11-20 nm spherical particles containing 70% of their cholesterol in the esterifi ed form, with a similar density to ␣ -HDL found in plasma ( 38,(40)(41)(42). Plasma HDL maturation is catalyzed by LCAT, which esterifi es free cholesterol (FC) to cholesteryl ester (CE) to form the lipid core critical for conversion of discoidal pre ␤ -HDL to mature spherical ␣ -HDL ( 43 ). In addition to HDL, LCAT can also esterify FC contained on other lipoprotein particles, thus infl uencing their metabolism as well. In plasma, HDL maturation maintains the gradient of FC between the cell membrane and HDL surface, thereby driving cholesterol effl ux, a key process in reverse cholesterol transport ( 43 ). containing complete protease inhibitor (Roche Applied Science) in a Tissuemite homogenizer for 20 s, sonicated at 20% output for 10 s, and clarifi ed by centrifugation at 16,600 rcf for 45 min at 4°C. The supernatant (carbonate-soluble fraction) was removed and neutralized by adding approximately 1.5 volumes of 1 M Tris (pH 6.8) to give a fi nal pH of approximately 7.4. The remaining pellet was resuspended in guanidine hydrochloride (GuHCl) extraction buffer [5 M GuHCl, 50 mM Tris HCl (pH 7.4)]. For measurement of LCAT, cortex and liver samples were homogenized in 8 volumes of ice-cold RIPA lysis buffer containing complete protease inhibitor (Roche Applied Science) in a Tissuemite homogenizer for 20 s, sonicated at 20% output for 10 s, and centrifuged at 8,600 rcf for 10 min at 4°C. Protein concentrations were determined using a BCA assay (Pierce).

A ␤ ELISA
Human A ␤ 40 and A ␤ 42 levels were quantifi ed by commercial ELISA kits (Invitrogen, KMB3482 and KMB3442) following the manufacturer's instructions. Levels of A ␤ 40 and A ␤ 42 were normalized to total protein.

Histology
Five 25 m sagittal sections spaced approximately 300 M apart spanning the entire length of the hippocampus were analyzed per animal. Sections were mounted on SuperFrost Plus glass slides (Fisher Scientifi c), immersed for 10 min in 1% thiofl avin S solution, washed with water, and coverslipped using Vectashield Hard Set mounting medium (Vector Laboratories). An additional two sections per mouse were fi rst stained with resorufi n, which selectively binds vascular amyloid ( 54 ). Sections were washed with PBS, permeabilized for 30 min in PBS containing 0.25% Triton X-100 (PBS-T), stained with 1 M resorufi n in PBS-T for 30 min, followed by three washes in PBS, one wash in 1:1 PBS and ethanol, and three washes in PBS. Sections were then mounted and stained with thiofl avin S as described above. Images were taken using a BX61 fl uorescent microscope (Olympus) and quantifi ed using Image Pro (Media Cybernetics) software. Amyloid within the area of interest (cortex, hippocampus, thalamus, or cortical vessels) was identifi ed by intensity-level threshold. Amyloid load (defi ned as the sum of thiofl avin S positive area /total area analyzed × 100) was calculated for each section and then averaged across sections for each individual mouse. Total amyloid (parenchymal + vascular) was measured using thiofl avin S while vascular amyloid in the cortex was measured using both thiofl avin S and resorufi n.
(Bimeda-MTC). CSF was isolated from the cisterna magna as described ( 49 ). Blood was collected via cardiac puncture, placed into tubes containing EDTA, and centrifuged at 21,000 rcf for 10 min and stored at Ϫ 80°C until use. Animals were then perfused for 7 min at 8 ml/min with PBS containing 2,500 U/l heparin. Liver and brain were extracted and the cortex and hippocampus were dissected and snap-frozen separately at Ϫ 80°C. Half of the brain was placed into 10% neutral buffered formalin followed by 30% sucrose in PBS for histological analysis.

Plasma lipid and lipoprotein analysis
Plasma HDL cholesterol (HDL-C) was determined using commercially available kits (Wako Diagnostics) according to the manufacturer's instructions. Plasma lipoprotein species were further analyzed by separating them based on size and charge and staining for lipids using Sudan Black. Hydragel 15 Lipoprotein(E) kits (Sebia, 4134) were purchased from Sebia, Inc. Neat plasma (10 l) was loaded into each well of the applicator and left at room temperature for 5 min. A Hydragel 15 Lipoprotein(E) gel was quickly blotted with thin fi lter paper to absorb excess liquid on the surface of the gel according to manufacturer's instructions. After the 5 min loading period, the applicator was placed perpendicular to the Hydragel, 3-4 cm from the bottom of the gel. After a 10 min application of sample to the gel, the applicator was removed and the gel was subjected to electrophoresis in 1× barbital buffer (Sigma, B5934-12VL, 1 vial dissolved in 1 l distilled water) in a Titan gel electrophoresis apparatus (Helena Laboratories) with 25 ml barbital buffer per side. The gel was run for 40 min at 100 V using a Bio-Rad PowerPac 100 power supply. After the run, the gel was placed in fi xing solution (3% methanol, 57% ethanol, and 10% glacial acetic acid in distilled water) for 10 min. The gel was then dried in a 60°C oven (3 h to overnight), incubated in staining solution (54% ethanol in distilled water containing 200 l Sudan black per 40 ml fi nal volume) for 10-15 min, destained in discoloring solution (45% ethanol in distilled water) for 5 min, and then dried in a 60°C oven for 10 min.

Plasma LCAT activity assay
LCAT activity in plasma was assayed as described previously ( 50 ) with modifi cations ( 51 ). Briefl y, artifi cial substrate for LCAT, [1,[2][3] H(N)]cholesterol (Perkin Elmer, NET139001MC) labeled liposomes were prepared as described ( 51 ). Each plasma sample was analyzed in triplicate using 2 l per measurement. Tubes with 60 l of labeled liposomes and 2 l of plasma were incubated for 30 min at 37°C, after which the reaction was stopped by 1 ml of cold ethanol. Tubes were kept at Ϫ 20°C for 2-12 h and then centrifuged at 13,000 g for 10 min at 4°C. The supernatant was evaporated in a SpeedVac and then dissolved in 30 l of chloroform with addition of unlabeled cholesterol and CE markers. The substrate and product of the LCAT reaction were separated by thin-layer chromatography using fl exible silica polyester backed plates (Whatman, 4410221). The FC and cholesteryl oleate spots on the plates were localized by standard staining in the chamber with iodine vapor, then transferred into scintillation vials and counted. The results obtained were used to calculate the coeffi cient of esterifi cation (ratio CE/FC) and specifi c LCAT activity in nanomoles per milliliter per hour. In these calculations, the original amount of FC in the reaction was calculated as the cholesterol in liposomes plus FC taken with 2 l of plasma ( 52 ).

RESULTS
LCAT expression and activity in plasma is not affected by aging or the onset of amyloid pathology in APP/PS1 mice As there has been a single report of decreased CSF LCAT activity in human AD subjects ( 42 ), we fi rst determined whether age and/or the presence of A ␤ deposits impacts LCAT expression or activity in APP/PS1 mice. As amyloid deposition and behavioral defi cits have been reported to start between 6 and 8 months of age in this line, we analyzed male APP/PS1 mice at 5 months of age, prior to disease onset, at 11 months of age, where mice demonstrate moderate pathology, and at 15.5 months of age, the age of the mice used in this study when pathology is very pronounced. Neither liver nor cortical LCAT mRNA was signifi cantly changed with age ( Fig. 1A ). We next measured the total circulating levels of plasma LCAT and apoA-I, which is the major physiological activator of LCAT in plasma. There was a signifi cant 27% increase in plasma LCAT levels and a nonsignifi cant 36% increase of plasma apoA-I in 15.5-month-old APP/PS1 mice compared with 5-month-old APP/PS1 mice ( Fig. 1B, C ). However, plasma LCAT activity, measured directly using artifi cial liposome substrates ex vivo ( Fig. 1D ) and measured indirectly by quantifying the relative abundance of plasma mRNA extraction and quantitative RT-PCR Liver and brain cortex tissue were homogenized in Trizol with a Precellys24 homogenizer and RNA was extracted and purifi ed with a PureLink mini kit followed by quantifi cation using a nanodrop spectrophotometer at 260 nm. Absorbance at 230 and 280 nm was also measured to determine nucleic acid purity. One microgram of RNA was reverse-transcribed in cDNA by TaqMan reverse transcriptase reagents kit (Life Technologies) and 40 ng of cDNA was used for each quantitative PCR reaction. LCAT gene expression in liver and brain cortex was evaluated through realtime PCR assay by the TaqMan method. Mm00500505_m1 probe (Life Technologies) was utilized for LCAT evaluation and ␤ -actin was utilized as housekeeping gene (Life Technologies, 4352341E probe). Data were calculated by the comparative C T ( ⌬ ⌬ C T ) method with normalization of the raw data to ␤ -actin and expressed as fold-change relative to the 5-month-old APP/PS1 mice prior to amyloid deposition.

Statistical analysis
Data were analyzed by either an unpaired two-tailed Student's t -test or one-way ANOVA followed by a Bonferroni post hoc test for normally distributed data or a Mann Whitney test or Kruskal-Wallis test followed by a Dunn's multiple comparison test for nonparametric data. All statistical calculations were performed using GraphPad Prism v5.0. Fig. 1. Expression, levels, and plasma activity of LCAT are unaffected by age or the accumulation of A ␤ and amyloid. LCAT mRNA, protein levels, and activity in addition to plasma apoA-I levels were measured in 5-, 11-, and 15.5-month-old male APP/PS1 mice. Plasma from young LCAT Ϫ / Ϫ mice, denoted as simply Ϫ / Ϫ , was used as a reference control. LCAT mRNA in the liver and cortex of APP/PS1 mice was measured by qRT-PCR (A). B: Plasma LCAT protein levels were measured by denaturing immunoblotting. C: Plasma levels of apoA-I were measured by denaturing immunoblotting. D: The activity of plasma LCAT was measured by determining the coeffi cient of esterifi cation, defi ned by the ratio of CE/FC, following incubation of plasma with 3 H-cholesterol-labeled liposomes. E: Plasma ␣ HDL levels were quantifi ed as a secondary measure of LCAT activity. Plasma samples were subjected to native gel electrophoresis through Sebia gels and stained with Sudan black, which binds to lipids. Purifi ed human HDL and LDL were run for comparison. Graphs represent mean ± SEM with N = 3-10 per group. Data were analyzed using a Kruskal-Wallis test followed by a Dunn's multiple comparison test. brain tissue or CSF ( Fig. 3B ). Further, the size and distribution of apoE-containing lipoprotein particles in CSF were similar between APP/PS1 WT and APP/PS1 LCAT Ϫ / Ϫ mice (supplementary Fig. I). Lastly, we investigated whether LCAT defi ciency impacts CNS expression of key receptors involved in cholesterol and lipoprotein metabolism, namely ABCA1, LDLR, LRP1, and scavenger receptor BI (SR-BI) (supplementary Fig. II). The absence of LCAT has no effect on cortical and hippocampal levels of ABCA1 and LRP1 (supplementary Fig. IIA, C), but was associated with a 40% increase in hippocampal LDLR levels (supplementary Fig.  IIB; P = 0.0317) and a 71% decrease in hippocampal scavenger receptor BI (supplementary Fig. 2D; P = 0.0357).

A ␤ and amyloid deposition are not increased by loss of LCAT in APP/PS1 mice
If LCAT-mediated lipoprotein maturation is required for effi cient A ␤ clearance, LCAT defi ciency would be expected to elevate A ␤ levels and amyloid burden in vivo. We observed no consistent and signifi cant changes in either soluble or aggregated A ␤ levels and in total or vascular amyloid burden ( Figs. 4, 5 ). In contrast to the expected observation of increased A ␤ levels, we were surprised to fi nd a 46% decrease ( P = 0.0173) in cortical levels of carbonate soluble A ␤ 42 in APP/PS1 LCAT Ϫ / Ϫ mice, albeit this was the only signifi cant change detected for A ␤ levels ( Fig. 4 ). Notably, the ratio of A ␤ 40:A ␤ 42 was increased by 70% in carbonate and guanidine soluble cortical lysates ( P = 0.0357 carbonate soluble, P = 0.1181 GuHCl soluble) and by 130% in guanidine soluble hippocampal lysates ( P = 0.0087). Histological analysis using thiofl avin-A, which detects total amyloid, and resorufi n staining, which specifi cally binds to vascular amyloid ( 54 ), revealed no change in total or cerebrovascular amyloid burden between APP/ PS1 WT and APP/PS1 LCAT Ϫ / Ϫ mice ( Fig. 5 ).
␣ -migrating HDL ( Fig. 1E ), was unaffected by age or the onset of amyloid deposition. Although it would have been ideal to measure LCAT activity in the CSF of these animals, these experiments were not feasible due to the low yield of CSF and dilute LCAT levels in CSF.

LCAT defi ciency selectively reduces plasma and CNS apoA-I
Next, we analyzed plasma lipoprotein levels and distribution following total body deletion of LCAT from male APP/PS1 mice. As expected, plasma apoA-I and total HDL-C were dramatically reduced to 14% and 8% of APP/ PS1 WT littermate control levels ( Fig. 2A, B ). Plasma lipoprotein subspecies were further characterized by separating whole plasma using Sebia gels, which separate lipoprotein particles based on surface charge, followed by Sudan black staining to detect lipids. As expected, deletion of LCAT from APP/PS1 mice results in an almost complete absence of mature ␣ -migrating HDL and an enrichment of lipoproproteins at the gel origin, which most likely includes chylomicrons ( 43 ) ( Fig. 2C ). These results are consistent with previous characterization of young male LCAT Ϫ / Ϫ mice on a C57Bl.6 background ( 45 ), indicating that advanced age and the onset of amyloid pathology does not infl uence the robust plasma lipoprotein phenotype induced by LCAT defi ciency.
We then assessed the impact of LCAT defi ciency on the levels of apoA-I-and apoE-containing lipoprotein particles in the CNS. While apoA-I is considered the major physiological activator of LCAT in the plasma ( 43 ), this role is believed to be fi lled by apoE in the CNS ( 47 ). In parallel to the observations in plasma, the level of apoA -I in the cortex, hippocampus, and CSF of APP/PS1 LCAT Ϫ / Ϫ mice was reduced to 30% ( P < 0.0001), 10% ( P = 0.0571), and 11% of APP/PS1 WT littermate controls ( Fig. 3A ). Conversely, loss of LCAT did not affect apoE levels in either Fig. 2. Deletion of LCAT in APP/PS1 mice signifi cantly reduces HDL-C, specifi cally ␣ -migrating particles. A: Plasma apoA-I protein levels were measured by denaturing immunoblotting. B: Total plasma HDL-C was measured using a commercial enzymatic kit. C: Plasma samples were subjected to native gel electrophoresis through Sebia gels and stained with Sudan black, which binds to lipids. Purifi ed human HDL and LDL were run for comparison. Graphs represent mean ± SEM with N = 5-6 per group. Data were analyzed using Mann-Whitney test (A) and Student's unpaired t -test (B).
LCAT defi ciency may have altered the age of onset of A ␤ or amyloid deposition.
It is also important to note that both apoE and apoA-I lipoproteins may affect A ␤ and amyloid metabolism. Lefterov et al. ( 55 ) previously reported a robust and specifi c increase of cerebrovascular amyloid angiopathy in 12-month-old APP/ PS1 mice that were crossed onto a total apoA-I knockout background, suggesting that apoA-I specifi cally affects vascular amyloid levels. We observed no change in vascular amyloid burden in APP/PS1 LCAT Ϫ / Ϫ mice ( Fig. 5F ), despite a 70-90% reduction in plasma and CNS apoA-I ( Figs. 2A , 3A). However, LCAT-defi cient mice are not equivalent to apoA-Idefi cient mice, as approximately 10% of apoA-I remains in the absence of LCAT and much of this apoA-I is believed to be in the pre ␤ form ( 44,45 ). It is therefore possible that pre ␤ apoA-I HDL is more important or more effi cient than ␣ -HDL with respect to amyloid clearance within the cerebrovasculature. Although not specifi cally measured in our study, there have been previous reports of increased plasma pre ␤ HDL in LCAT-defi cient mice ( 44,45 ) and in human subjects with DISCUSSION Although LCAT is well-characterized with respect to its impact on peripheral HDL metabolism ( 43 ), little is known about its function in the CNS. While the initial lipidation of glial-derived apoE by ABCA1 is a critical determinant in its ability to mediate A ␤ degradation and clearance ( 32,33,35 ), it is unknown whether the generation of the CE core by LCAT is also required. Our data show that total body deletion of LCAT, which blocks HDL lipoprotein maturation, does not signifi cantly impair A ␤ or amyloid deposition in APP/PS1 mice ( Fig. 4, 5 ). This fi nding provides strong support for the hypothesis that A ␤ clearance is mediated by nascent discoidal lipoprotein particles (whether apoA-I or apoE derived) rather than mature spherical plasma or CNS lipoproteins. As APP/PS1 mice develop vascular amyloid deposits much later than parenchymal deposits, we specifically designed our study to investigate A ␤ and amyloid load at 15.5 months of age, when both total and vascular deposits are robust. However, we cannot rule out the possibility that  tangles or marked neurodegeneration, they may not model all of the components of AD pathology that may act in concert to suppress LCAT activity in human CSF. Although LCAT defi ciency does not impair A ␤ metabolism in APP/PS1 mice, it is important to recognize that LCAT may have other important functions with respect to CNS lipoprotein metabolism. Lipoprotein maturation is a key step in lipoprotein-mediated transfer of lipids from one tissue source to another through body fl uids. As apoEcontaining lipoproteins are the major source by which adult neurons acquire the lipids necessary for the maintenance and repair of membranes, it is possible that the impact of LCAT defi ciency may be most evident when apoE is required for lipid transport, such as under acute conditions of neuronal damage. Traumatic brain injury in humans, for example, leads to a signifi cant decrease in CSF apoE levels but not of CSF apoA-I levels ( 57 ), and loss of apoE impairs recovery after traumatic brain injury in mice ( 58 ). Future studies may reveal a role for CNS LCAT to facilitate lipid transport in response to acute neuronal damage. mutant LCAT alleles resulting in increased ABCA1-mediated cholesterol effl ux ( 56 ). In contrast to apoA-I, the electrophoretic mobility characteristics of nascent discoidal apoE particles is not known.
Another fi nding of this study is that neither age nor the presence of amyloid deposits affects LCAT expression in the liver and cortex, or more importantly, plasma LCAT activity in the APP/PS1 mouse model ( Fig. 1 ). In contrast, a 50% decrease in CSF LCAT activity in human AD subjects compared with age-matched nondemented controls has been previously reported ( 42 ). Although it would be ideal to measure LCAT activity in the CSF of APP/PS1 mice, the small volume of CSF (approximately 10 l per mouse) obtained from mice and predicted LCAT levels and activity (2.5% of that in serum) pose signifi cant challenges to this experiment ( 42 ). Future studies will be needed to determine if CSF LCAT levels are consistently reduced in AD subjects and, if so, whether this is correlated with other AD-relevant CSF biomarker changes, including A ␤ and tau. As APP/PS1 mice do not develop neurofi brillary