Cholesterol Clearance Research Articles

Familial hypercholesterolaemia workshop for leveraging point-of-care testing and personalised medicine in association with the Lipid and Atherosclerosis Society of Southern Africa.

Familial hypercholesterolaemia (FH) is a common autosomal dominantly inherited disorder in which impaired clearance of plasma low-density lipoprotein cholesterol causes premature atherosclerotic vascular disease and tendon xanthomata. This workshop aimed to consolidate information on the diagnosis and management of FH in South Africa. The genetic causes include mutations in the LDL receptor, apolipoprotein B100 and proprotein convertase subtilisin/kexin type 9 (PCSK9). Additionally, the concatenation of multiple gene variants can result in polygenic FH. Therapeutic measures include a healthy lifestyle, statins and cholesterol-absorption inhibitors that will achieve control of the dyslipidaemia in the majority of cases. The recently introduced monoclonal antibodies to PCSK9 can improve achievement of target concentration in severe cases. FH is present in all sectors of the South African population but there is sparse documentation in the indigenous African populations. FH should be actively sought, diagnosed and treated with judicious pharmacotherapy and screening of relatives.

Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test.

Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous, multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study was to establish a test that allows for an objective assessment of the pathological potential of NPC1 gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying distinct sequence variants. Filipin staining was used to test for complementation of the phenotype. The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and 24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met, and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance. Moreover, we present a system that can be utilized to screen for new drugs.

Loss of spatacsin impairs cholesterol trafficking and calcium homeostasis

Mutations in SPG11, leading to loss of spatacsin function, impair the formation of membrane tubules in lysosomes and cause lysosomal lipid accumulation. However, the full nature of lipids accumulating in lysosomes and the physiological consequences of such accumulation are unknown. Here we show that loss of spatacsin inhibits the formation of tubules on lysosomes and prevents the clearance of cholesterol from this subcellular compartment. Accumulation of cholesterol in lysosomes decreases cholesterol levels in the plasma membrane, enhancing the entry of extracellular calcium by store-operated calcium entry and increasing resting cytosolic calcium levels. Higher cytosolic calcium levels promote the nuclear translocation of the master regulator of lysosomes TFEB, preventing the formation of tubules and the clearance of cholesterol from lysosomes. Our work reveals a homeostatic balance between cholesterol trafficking and cytosolic calcium levels and shows that loss of spatacsin impairs this homeostatic equilibrium.

Intracellular cholesterol trafficking is dependent upon NPC2 interaction with lysobisphosphatidic acid.

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein.

MiRNA-Gene Regulatory Network in Gnotobiotic Mice Stimulated by Dysbiotic Gut Microbiota Transplanted From a Genetically Obese Child.

Gut microbiota (GM) dysbiosis has been considered a pathogenic origin of many chronic diseases. In our previous trial, a shift in GM structure caused by a complex fiber-rich diet was associated with the health improvement of obese Prader-Willi syndrome (PWS) children. The pre- and post-intervention GMs (pre- and post-group, respectively) from one child were then transplanted into gnotobiotic mice, which resulted in significantly different physiological phenotypes, each of which was similar to the phenotype of the corresponding GM donor. This study was designed to investigate the miRNA-gene regulatory networks involved in causing these phenotypic differences. Using the post-group as a reference, we systematically identified and annotated the differentially expressed (DE) miRNAs and genes in the colon and liver of the pre-group in the second and fourth weeks after GM inoculation. Most of the significantly enriched GO terms and KEGG pathways were observed in the liver and were in the second week after GM transplantation. We screened 23 key genes along with their 73 miRNA regulators relevant to the host phenotype changes and constructed a network. The network contained 92 miRNA-gene regulation relationships, 51 of which were positive, and 41 of which were negative. Both the colon and liver had upregulated pro-inflammatory genes, and genes involved in fatty acid oxidation, lipolysis, and plasma cholesterol clearance were downregulated in only the liver. These changes were consistent with lipid and cholesterol accumulation in the host and with a high inflammation level. In addition, the colon showed an impacted glucagon-like peptide 1 (GLP-1) signaling pathway, while the liver displayed decreased insulin receptor signaling pathway activity. These molecular changes were mainly found in the second week, 2 weeks before changes in body fat occurred. This time lag indicated that GM dysbiosis might initially induce cholesterol and lipid metabolism-related miRNA and gene expression disorder and then lead to lipid accumulation and obesity development, which implicates a causative role of GM dysbiosis in obesity development rather than a result of obesity. This study provides fundamental molecular information that elucidates how dysbiotic GM increases host inflammation and disturbs host lipid and glucose metabolism.

Effects of dietary ascorbic acid levels on cholesterol metabolism in rainbow trout (Oncorhynchus mykiss)

An 80‐day feeding trial was conducted to evaluate the effects of dietary ascorbic acid (AA) supplementation at different levels (0, 0.2 and 0.4 g/kg l‐ascorbate‐2‐polyphosphate; 7.6, 77.2 and 146.7 mg/kg AA, respectively) on cholesterol metabolism in rainbow trout (Oncorhynchus mykiss). Dietary AA supplementation regardless of inclusion level increased the serum total cholesterol and low‐density lipoprotein (LDL) cholesterol levels and LDL cholesterol/high‐density lipoprotein cholesterol ratio. No significant differences were observed in the serum triiodothyronine and thyroxine levels, faecal cholesterol content, hepatic activity and mRNA expression of acyl‐coenzyme A:cholesterol acyltransferase among the dietary treatments. Dietary AA inclusion increased the faecal bile acid content, hepatic activity and mRNA expression of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and hepatic cholesterol 7α‐hydroxylase mRNA expression, but decreased the hepatic LDL receptor content. High level of AA supplementation (0.4 g/kg) depressed the serum cortisol levels. These results suggest that dietary supplementation with 0.2‒0.4 g/kg l‐ascorbate‐2‐polyphosphate may increase the serum total cholesterol level in rainbow trout. The cholesterol‐raising effect of AA may be due to the increased hepatic cholesterol production and the depressed cholesterol clearance from serum. In addition, dietary AA inclusion also facilitates the hepatic conversion of cholesterol to bile acids.

The hepatic WASH complex is required for efficient plasma LDL and HDL cholesterol clearance

The evolutionary conserved Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex is one of the crucial multiprotein complexes that facilitates endosomal recycling of transmembrane proteins. Defects in WASH components have been associated with inherited developmental and neurological disorders in humans. Here, we show that hepatic ablation of the WASH component Washc1 in chow-fed mice increases plasma concentrations of cholesterol in both LDLs and HDLs, without affecting hepatic cholesterol content, hepatic cholesterol synthesis, biliary cholesterol excretion, or hepatic bile acid metabolism. Elevated plasma LDL cholesterol was related to reduced hepatocytic surface levels of the LDL receptor (LDLR) and the LDLR-related protein LRP1. Hepatic WASH ablation also reduced the surface levels of scavenger receptor class B type I and, concomitantly, selective uptake of HDL cholesterol into the liver. Furthermore, we found that WASHC1 deficiency increases LDLR proteolysis by the inducible degrader of LDLR, but does not affect proprotein convertase subtilisin/kexin type 9-mediated LDLR degradation. Remarkably, however, loss of hepatic WASHC1 may sensitize LDLR for proprotein convertase subtilisin/kexin type 9-induced degradation. Altogether, these findings identify the WASH complex as a regulator of LDL as well as HDL metabolism and provide in vivo evidence for endosomal trafficking of scavenger receptor class B type I in hepatocytes.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

Real-World Outcomes with Lomitapide Use in Paediatric Patients with Homozygous Familial Hypercholesterolaemia.

IntroductionHomozygous familial hypercholesterolaemia (HoFH) is a rare, autosomal disease affecting the clearance of low-density lipoprotein cholesterol (LDL-C) from circulation, and leading to early-onset atherosclerotic cardiovascular disease (ASCVD). Treatment consists mainly of statins, lipoprotein apheresis (LA) and, more recently, the microsomal triglyceride transfer protein inhibitor lomitapide. Lomitapide is not licensed for use in children, but has been made available through an expanded access programme or on a named patient basis.MethodsThis case series includes 11 HoFH patients in 10 different centres in eight countries, less than 18 years of age (mean 11.6 ± 1.1 years, 64% male), with signs of ASCVD, and who have received treatment with lomitapide (mean dose 24.5 ± 4.3 mg/day; mean exposure 20.0 ± 2.9 months). Background lipid-lowering therapy was given according to local protocols. Lomitapide was commenced with a stepwise dose escalation from 2.5 mg or 5 mg/day; dietary advice and vitamin supplements were provided as per the product label for adults. Laboratory analysis was conducted as part of regular clinical care.ResultsIn the 11 cases, mean baseline LDL-C was 419 ± 74.6 mg/dL and was markedly reduced by lomitapide to a nadir of 176.7 ± 46.3 mg/dL (58.4 ± 6.8% decrease). Six patients achieved recommended target levels for children below 135 mg/dL, five of whom had LA frequency reduced. In one case, LDL-C levels were close to target when lomitapide was started but remained stable despite 75% reduction in LA frequency (from twice weekly to biweekly). Adverse events were mainly gastrointestinal in nature, occurred early in the treatment course and were well managed. Three patients with excursions in liver function tests were managed chiefly without intervention; two patients had decreases in lomitapide dose.ConclusionsLomitapide demonstrated promising effectiveness in paediatric HoFH patients. Adverse events were manageable, and the clinical profile of the drug is apparently similar to that in adult patients.FundingAmryt Pharma.

HDL subclass proteomic analysis and functional implication of protein dynamic change during HDL maturation

Recent clinical trials reported that increasing high-density lipoprotein-cholesterol (HDL-C) levels does not improve cardiovascular outcomes. We hypothesize that HDL proteome dynamics determine HDL cardioprotective functions. In this study, we characterized proteome profiles in HDL subclasses and established their functional connection. Mouse plasma was fractionized by fast protein liquid chromatography, examined for protein, cholesterial, phospholipid and trigliceride content. Small, medium and large (S/M/L)-HDL subclasseses were collected for proteomic analysis by mass spectrometry. Fifty-one HDL proteins (39 in S-HDL, 27 in M-HDL and 29 in L-HDL) were identified and grouped into 4 functional categories (lipid metabolism, immune response, coagulation, and others). Eleven HDL common proteins were identified in all HDL subclasses. Sixteen, 3 and 7 proteins were found only in S-HDL, M-HDL and L-HDL, respectively. We established HDL protein dynamic distribution in S/M/L-HDL and developed a model of protein composition change during HDL maturation. We found that cholesterol efflux and immune response are essential functions for all HDL particles, and amino acid metabolism is a special function of S-HDL, whereas anti-coagulation is special for M-HDL. Pon1 is recruited into M/L-HDL to provide its antioxidative function. ApoE is incorporated into L-HDL to optimize its cholesterial clearance function. Next, we acquired HDL proteome data from Pubmed and identified 12 replicated proteins in human and mouse HDL particle. Finally, we extracted 3 shared top moleccular pathways (LXR/RXR, FXR/RXR and acute phase response) for all HDL particles and 5 top disease/bio-functions differentially related to S/M/L-HDL subclasses, and presented one top net works for each HDL subclass. We conclude that beside their essencial functions of cholesterol efflux and immune response, HDL aquired antioxidative and cholesterol clearance functions by recruiting Pon1 and ApoE during HDL maturation.

Targeting Molecular Chaperone HSP90 To Treat Niemann‐Pick Type C1 Disease

NPC1 is a fatal, neurodegenerative lysosomal storage disease caused by mutations in the NPC1 gene. More than 300 different disease‐associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids and other lipids in late endosomes/lysosomes (LE/Ly) of many cell types. There is no FDA‐approved therapy to treat NPC1 disease. A limited initial human trial of hydroxylpropyl beta cyclodextrin (HPBCD) is currently in progress. Our previous studies suggested that treatment with histone deacetylase inhibitors (HDACis) on skin fibroblasts results in increased expression of the NPC1 protein in mutant cells. Using an engineered human cell line, we have observed that HDACis can correct the NPC1 cholesterol storage defect in 60 of the 81 NPC1 mutants tested. However, when HDACis', were tested on non‐dividing cells like bone marrow derived macrophages, we did not observe any correction.Although, significant progress has been made in understanding the pathology of NPC1 disease, the mechanisms governing the proteostatic control of NPC1 protein is not well understood.. Disruption of molecular chaperone HSP90 (via hyperacetylation) is one of the many consequences of HDACi treatment. Thus, we evaluated the effect of direct inhibition of HSP90 family of proteins on NPC1 skin fibroblasts in terms of lowering lysosomal cholesterol accumulation.The data on the effect of several different HSP90 inhibitors on human patient‐derived skin fibroblast in lowering lysosomal cholesterol burden using microscopy based filipin assay will be presented. Our results suggests that cytosolic HSP90 and associated chaperones may be a target and that protein folding quality control plays a critical role in NPC1 diseases. We observed that out of the three HSP90 family proteins, inhibiting HSP90A and not HSP90B or TRAP resulted in clearance of cholesterol from lysosomes as well as aided in mobilizing unfolded mutant NPC1 protein from ER to lysosomes. We will also present the results elucidating the role of cytosolic HSP90 and its associated co‐chaperones in the regulation of NPC1 mutant protein stability.Support or Funding Information1. MDBR‐Orphan Disease UPenn Grant to Dr. Pipalia 2. APMRF Grant to Dr. Maxfield3. DART Summer Fellowship to Abigail CrossThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Eicosapentaenoic acid and docosahexaenoic acid, but not α-linolenic acid, decreased low-density lipoprotein cholesterol synergistically with estrogen via regulation of cholesterol synthesis and clearance in ovariectomized rats

Our previous study showed that n-3 polyunsaturated fatty acid (PUFA) and estrogen (E) had synergistic hypocholesterolemic effects by inhibiting cholesterol synthesis and enhancing bile acid synthesis. The purpose of the present study was to investigate the hypothesis that α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) decrease low-density lipoprotein cholesterol (LDL-C), synergistically with E, via hepatic cholesterol synthesis and clearance. Rats were fed a diet with either 0% n-3 PUFA or 1% ALA, EPA, or DHA, relative to total energy consumption, for the entire 12-week study. After ovariectomy, rats were injected with either corn oil or E every 4 days for the last 3 weeks of the study. In combination with E, dietary supplementation with EPA or DHA increased the phosphorylated adenosine monophosphate–activated protein kinase/adenosine monophosphate–activated protein kinase ratio and LDL receptor expression, and it decreased the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, sterol regulatory element-binding protein-2, and proprotein convertase subtilisin/kexin type 9 in the liver. In addition, dietary supplementation with EPA or DHA increased hepatic expression of cholesterol 7α-hydroxylase, sterol 12α-hydroxylase, and sterol 27-hydroxylase. However, E decreased the expression of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase and increased the expression of estrogen receptor α and β in the liver. ALA had no significant effects on cholesterol metabolism. In conclusion, the present study suggests that dietary supplementation with EPA and DHA decreased LDL-C synthesis and increased bile acid synthesis and LDL-C clearance by LDL receptor, synergistically with E.

Pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by the accumulation of alveolar surfactant and dysfunction of alveolar macrophages. PAP results in progressive dyspnoea of insidious onset, hypoxaemic respiratory failure, secondary infections and pulmonary fibrosis. PAP can be classified into different types on the basis of the pathogenetic mechanism: primary PAP is characterized by the disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling and can be autoimmune (caused by elevated levels of GM-CSF autoantibodies) or hereditary (due to mutations in CSF2RA or CSF2RB, encoding GM-CSF receptor subunits); secondary PAP results from various underlying conditions; and congenital PAP is caused by mutations in genes involved in surfactant production. In most patients, pathogenesis is driven by reduced GM-CSF-dependent cholesterol clearance in alveolar macrophages, which impairs alveolar surfactant clearance. PAP has a prevalence of at least 7 cases per million individuals in large population studies and affects men, women and children of all ages, ethnicities and geographical locations irrespective of socioeconomic status, although it is more-prevalent in smokers. Autoimmune PAP accounts for >90% of all cases. Management aims at improving symptoms and quality of life; whole-lung lavage effectively removes excessive surfactant. Novel pathogenesis-based therapies are in development, targeting GM-CSF signalling, immune modulation and cholesterol homeostasis.

Oat fiber inhibits atherosclerotic progression through improving lipid metabolism in ApoE−/− mice

Cardiovascular diseases caused by atherosclerosis have become one of the leading cause of death. The aim of the present study was to explore how oat fiber regulated lipid metabolism and to elucidate its anti-atherosclerotic potential in ApoE−/− mice. Animals were daily fed a high-fat diet without or with 0.8% oat fiber for 18 weeks. Both serum total cholesterol and low-density cholesterol levels decreased in mice received oat fiber administration than those without Oat fiber inhibited hepatic lipid accumulation by downregulating SREBP-1 expression and accelerated clearance of intestinal cholesterol by upregulating SREBP-2 expression. In addition, oat fiber activated cholesterol sensors LXRα, and then strengthened hepatic and intestinal cholesterol efflux by increasing ATP-binding cassette A1 and G1, and decreased intestinal cholesterol absorption by inhibiting Niemann pick C 1 like 1. The findings implicated that oat fiber had an anti-atherosclerotic potential via mechanisms of activating the SREBPs/LXRα pathway and improving lipid metabolism.

Cereal fiber improves blood cholesterol profiles and modulates intestinal cholesterol metabolism in C57BL/6 mice fed a high-fat, high-cholesterol diet.

BackgroundDietary intake of cereal fiber has been reported to benefit lipid metabolism through multiple mechanisms. The present study aimed to discover the potential mechanisms by which cereal fiber could modify the intestinal cholesterol metabolism.DesignMale C57BL/6 mice were fed a reference chow (RC) diet; high-fat, high-cholesterol (HFC) diet; HFC plus oat fiber diet; or HFC plus wheat bran fiber diet for 24 weeks. Serum lipids were measured by enzymatic methods. Western blot was used to determine the protein expressions involved in intestinal cholesterol metabolism.ResultsOur results showed that HFC-induced elevations of serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol were normalized in both groups that received cereal fiber. At the protein level, compared with the HFC diet group, the two cereal fibers, especially the oat fiber, significantly increased the protein expression of peroxisome proliferator-activated receptor alpha, liver X receptor alpha, sterol regulatory element-binding protein (SREBP) 2, low-density lipoprotein receptor, adenosine triphosphate (ATP)-binding cassette A1, and ATP-binding cassette G1, while decreasing the protein expression of Niemann-Pick C1-like protein 1, SREBP-1, fatty acid synthase, and acetyl-coenzyme A carboxylase, which were involved in intestinal cholesterol metabolism.ConclusionTaken together, increased intake of cereal fiber improved blood cholesterol profiles and increased the intestinal cholesterol efflux and cholesterol clearance in C57BL/6 mice fed a HFC diet. Oat fiber had a stronger effect than wheat bran fiber on cholesterol metabolism by modulating the PPARα, LXRα, and SREBP signaling pathways.

Diagnosis and management of children and young people with familial hypercholesterolaemia

Heterozygous familial hypercholesterolaemia is a common genetic condition characterised by the impaired clearance of low density lipoprotein cholesterol from the bloodstream. Incidence rates can be as high as 1/250 and those confirmed with familial hypercholesterolaemia are predisposed to progressive atherosclerosis throughout childhood, increasing their risk of premature cardiovascular disease. Early identification and initiation of risk factor management and lipid-lowering therapy are fundamental if this risk and its associated comorbidities are to be reduced and a life expectancy similar to that of the general population restored. This article discusses several aspects of familial hypercholesterolaemia including identification, diagnosis through genetic testing and treatment protocols in a tertiary cardiac centre. It aims to share key learning points with other institutions either already established in the care of paediatric patients with familial hypercholesterolaemia or those contemplating forming a service to meet the needs of this growing cohort.

Altered bile acid profile associates with cognitive impairment in Alzheimer's disease—An emerging role for gut microbiome

IntroductionIncreasing evidence suggests a role for the gut microbiome in central nervous system disorders and a specific role for the gut-brain axis in neurodegeneration. Bile acids (BAs), products of cholesterol metabolism and clearance, are produced in the liver and are further metabolized by gut bacteria. They have major regulatory and signaling functions and seem dysregulated in Alzheimer's disease (AD). MethodsSerum levels of 15 primary and secondary BAs and their conjugated forms were measured in 1464 subjects including 370 cognitively normal older adults, 284 with early mild cognitive impairment, 505 with late mild cognitive impairment, and 305 AD cases enrolled in the AD Neuroimaging Initiative. We assessed associations of BA profiles including selected ratios with diagnosis, cognition, and AD-related genetic variants, adjusting for confounders and multiple testing. ResultsIn AD compared to cognitively normal older adults, we observed significantly lower serum concentrations of a primary BA (cholic acid [CA]) and increased levels of the bacterially produced, secondary BA, deoxycholic acid, and its glycine and taurine conjugated forms. An increased ratio of deoxycholic acid:CA, which reflects 7α-dehydroxylation of CA by gut bacteria, strongly associated with cognitive decline, a finding replicated in serum and brain samples in the Rush Religious Orders and Memory and Aging Project. Several genetic variants in immune response–related genes implicated in AD showed associations with BA profiles. DiscussionWe report for the first time an association between altered BA profile, genetic variants implicated in AD, and cognitive changes in disease using a large multicenter study. These findings warrant further investigation of gut dysbiosis and possible role of gut-liver-brain axis in the pathogenesis of AD.

Induction of obesity impairs reverse cholesterol transport in ob/ob mice

ObjectivesObesity is an independent risk factor for cardiovascular disease. Reverse cholesterol transport (RCT) is an important cardioprotective mechanism. This study aimed to investigate RCT changes in a murine model of obesity.MethodsOb/ob and control mice were injected with [3H]-cholesterol-labelled macrophages and cholesterol accumulation quantified after 48 h. Ex vivo, cholesterol efflux and uptake were determined in hepatic and adipose tissues.ResultsOb/ob mice had more labelled cholesterol in their plasma (86%, p<0.001), suggesting impaired RCT. SR-BI-mediated cholesterol efflux was elevated from ob/ob mice (serum, 33%; apoB-depleted plasma, 14%, p<0.01) and HDL-c were also higher (60%, p<0.01). Ex vivo it was found that cholesterol uptake was significantly lower into the livers and adipose tissue of ob/ob mice, compared to non-obese wildtype controls. Furthermore, ex vivo cholesterol efflux was reduced in ob/ob liver and adipose tissue towards apoA-I and HDL. Consistent with this, protein levels of SR-BI and ABCG1 were significantly lower in ob/ob hepatic and adipose tissue than in wildtype mice. Finally, labelled cholesterol concentrations were lower in ob/ob bile (67%, p<0.01) and faeces (76%, p<0.0001).ConclusionObesity causes impairment in RCT due to reduced plasma cholesterol uptake and efflux by hepatocytes and adipocytes. A reduction in the capacity for plasma cholesterol clearance may partly account for increased CVD risk with obesity.

Impaired monocyte cholesterol clearance initiates age-related retinal degeneration and vision loss.

Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is characterized by atrophic neurodegeneration or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis. Here, we demonstrate that targeted deletion of macrophage cholesterol ABC transporters A1 (ABCA1) and -G1 (ABCG1) leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration. These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy.

The Role of heparan sulfate proteoglycans in PCSK9-induced LDL receptor degradation

Aim: PCSK9 binds the LDL receptor (LDLR) and induces its degradation. The result is reduced clearance of plasma cholesterol, and inhibitors preventing the binding of PCSK9 to LDLR are of immense clinical interest. However, the affinity of PCSK9 for LDLR is low, and it is an enigma why PCSK9 targets LDLR in the liver, but not in other tissues, all suggesting the requirement of a liver-specific co-receptor. We have found that PCSK9 binds heparin, a mimic of Heparan Sulfate (HS) proteoglycans (PG), and have investigated the role of this interaction in PCSK9 biology.