Progression of chronic kidney disease in familial LCAT deficiency: a follow-up of the Italian cohort

Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.     

A robust all-atom model for LCAT generated by homology modeling

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A₂ (PLA₂) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA₂. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.     

遗传性LCAT缺陷症与动脉粥样硬化

卵磷脂：胆固醇酰基转移酶（lecithin：cholesterol acyltransferase,LCAT）参与胆固醇酯的合成并在高密度脂蛋白（high density lipoprotein,HDL）的代谢中起重要作用。遗传性LCAT缺陷症是一种以低HDL-胆固醇（HDL-C）为特点的罕见遗传疾病。近年来,LCAT在HDL-C代谢中以及在动脉粥样硬化发生和发展中的作用逐渐被本领域研究者所关注。本文就LCAT缺陷症的遗传学和生化学特点做一综述,重点阐述为何尽管HDL-C水平明显减低,LCAT突变携带者却并未发生早期动脉粥样硬化。     

Depletion in LpA-I:A-II particles enhances HDL-mediated endothelial protection in familial LCAT deficiency

The aim of this study was to evaluate the vasoprotective effects of HDL isolated from carriers of LCAT deficiency, which are characterized by a selective depletion of LpA-I:A-II particles and predominance of preβ migrating HDL. HDLs were isolated from LCAT-deficient carriers and tested in vitro for their capacity to promote NO production and to inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in cultured endothelial cells. HDLs from carriers were more effective than control HDLs in promoting eNOS activation with a gene-dose-dependent effect (P_Trend = 0.048). As a consequence, NO production induced by HDL from carriers was significantly higher than that promoted by control HDL (1.63 ± 0.24-fold vs. 1.34 ± 0.07-fold, P= 0.031). HDLs from carriers were also more effective than control HDLs in inhibiting the expression of VCAM-1 (homozygotes, 65.0 ± 8.6%; heterozygotes, 53.1 ± 7.2%; controls, 44.4 ± 4.1%; P_Trend = 0.0003). The increased efficiency of carrier HDL was likely due to the depletion in LpA-I:A-II particles. The in vitro findings might explain why carriers of LCAT deficiency showed flow-mediated vasodilation and plasma-soluble cell adhesion molecule concentrations comparable to controls, despite low HDL-cholesterol levels. These results indicate that selective depletion of apoA-II-containing HDL, as observed in carriers of LCAT deficiency, leads to an increased capacity of HDL to stimulate endothelial NO production, suggesting that changes in HDL apolipoprotein composition may be the target of therapeutic interventions designed to improve HDL functionality.     

The high-resolution crystal structure of human LCAT

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.     

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.     

LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function

In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.001) and complete HDL-cholesteryl ester deficiency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoid-producing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40–50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting (P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl ester deficiency in LCAT KO mice is associated with a 40–50% lower adrenal glucocorticoid output. These findings further highlight the important novel role for HDL as cholesterol donor for the synthesis of glucocorticoids by the adrenals.     

ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.     

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.     

Three-dimensional colocalization analysis of plasma-derived apolipoprotein B with amyloid plaques in APP/PS1 transgenic mice

Parenchymal accumulation of amyloid-beta (Aβ) is a hallmark pathological feature of Alzheimer’s disease. An emerging hypothesis is that blood-to-brain delivery of Aβ may increase with compromised blood–brain barrier integrity. In plasma, substantial Aβ is associated with triglyceride-rich lipoproteins (TRLs) secreted by the liver and intestine. Utilizing apolipoprotein B as an exclusive marker of hepatic and intestinal TRLs, here we show utilizing an highly sensitive 3-dimensional immuno-microscopy imaging technique, that in APP/PS1 amyloid transgenic mice, concomitant with substantially increased plasma Aβ, there is a significant colocalization of apolipoprotein B with cerebral amyloid plaque. The findings are consistent with the possibility that circulating lipoprotein-Aβ contributes to cerebral amyloidosis.     

Differential impact of hepatic deficiency and total body inhibition of MTP on cholesterol metabolism and RCT in mice

Because apoB-containing lipoproteins are pro-atherogenic and their secretion by liver and intestine largely depends on microsomal triglyceride transfer protein (MTP) activity, MTP inhibition strategies are actively pursued. How decreasing the secretion of apoB-containing lipoproteins affects intracellular rerouting of cholesterol is unclear. Therefore, the aim of the present study was to determine the effects of reducing either systemic or liver-specific MTP activity on cholesterol metabolism and reverse cholesterol transport (RCT) using a pharmacological MTP inhibitor or a genetic model, respectively. Plasma total cholesterol and triglyceride levels were decreased in both MTP inhibitor-treated and liver-specific MTP knockout (L-Mttp^−/−) mice (each P < 0.001). With both inhibition approaches, hepatic cholesterol as well as triglyceride content was consistently increased (each P < 0.001), while biliary cholesterol and bile acid secretion remained unchanged. A small but significant decrease in fecal bile acid excretion was observed in inhibitor-treated mice (P < 0.05), whereas fecal neutral sterol excretion was substantially increased by 75% (P < 0.001), conceivably due to decreased intestinal absorption. In contrast, in L-Mttp^−/− mice both fecal neutral sterol and bile acid excretion remained unchanged. However, while total RCT increased in inhibitor-treated mice (P < 0.01), it surprisingly decreased in L-Mttp^−/− mice (P < 0.05). These data demonstrate that: i) pharmacological MTP inhibition increases RCT, an effect that might provide additional clinical benefit of MTP inhibitors; and ii) decreasing hepatic MTP decreases RCT, pointing toward a potential contribution of hepatocyte-derived VLDLs to RCT.     

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

Contribution of genetic and dietary insulin resistance to Alzheimer phenotype in APP/PS1 transgenic mice

According to epidemiological studies, type‐2 diabetes increases the risk of Alzheimer’s disease. Here, we induced hyperglycaemia in mice overexpressing mutant amyloid precursor protein and presenilin‐1 (APdE9) either by cross‐breeding them with pancreatic insulin‐like growth factor 2 (IGF‐2) overexpressing mice or by feeding them with high‐fat diet. Glucose and insulin tolerance tests revealed significant hyperglycaemia in mice overexpressing IGF‐2, which was exacerbated by high‐fat diet. However, sustained hyperinsulinaemia and insulin resistance were observed only in mice co‐expressing IGF‐2 and APdE9 without correlation to insulin levels in brain. In behavioural tests in aged mice, APdE9 was associated with poor spatial learning and the combination of IGF‐2 and high‐fat diet further impaired learning. Neither high‐fat diet nor IGF‐2 increased β‐amyloid burden in the brain. In male mice, IGF‐2 increased β‐amyloid 42/40 ratio, which correlated with poor spatial learning. In contrast, inhibitory phosphorylation of glycogen synthase kinase 3β, which correlated with good spatial learning, was increased in APdE9 and IGF‐2 female mice on standard diet, but not on high‐fat diet. Interestingly, high‐fat diet altered τ isoform expression and increased phosphorylation of τ at Ser202 site in female mice regardless of genotype. These findings provide evidence for new regulatory mechanisms that link type‐2 diabetes and Alzheimer pathology.     

Estrogen receptor α promotes Cav1.2 ubiquitination and degradation in neuronal cells and in APP/PS1 mice

Cav1.2 is the pore‐forming subunit of L‐type voltage‐gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen‐mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17β‐estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin–proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29‐deubiquitinating enzyme TRAF‐binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane‐permeable PEST peptides prevented ERα‐mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα‐induced Cav1.2 degradation involved K29‐linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.     

Clathrin adaptor AP-1–mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments

One of the hallmarks of Alzheimer’s disease is the accumulation of toxic amyloid-β (Aβ) peptides in extracellular plaques. The direct precursor of Aβ is the carboxyl-terminal fragment β (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer’s disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aβ. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aβ release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.