Tracing the lipidome in inborn errors of metabolism.

Inborn errors of metabolism (IEM) represent a heterogeneous group of more than 1800 rare disorders, many of which are causing significant childhood morbidity and mortality. More than 100 IEM are linked to dyslipidaemia, but yet our knowledge in connecting genetic information with lipidomic data is limited. Stable isotope tracing studies of the lipid metabolism (STL) provide insights on the dynamic of cellular lipid processes and could thereby facilitate the delineation of underlying metabolic (patho)mechanisms. This mini-review focuses on principles as well as technical limitations of STL and describes potential clinical applications by discussing recently published STL focusing on IEM.

Lipidomics-Paving the Road towards Better Insight and Precision Medicine in Rare Metabolic Diseases.

Even though the application of Next-Generation Sequencing (NGS) has significantly facilitated the identification of disease-associated mutations, the diagnostic rate of rare diseases is still below 50%. This causes a diagnostic odyssey and prevents specific treatment, as well as genetic counseling for further family planning. Increasing the diagnostic rate and reducing the time to diagnosis in children with unclear disease are crucial for a better patient outcome and improvement of quality of life. In many cases, NGS reveals variants of unknown significance (VUS) that need further investigations. The delineation of novel (lipid) biomarkers is not only crucial to prove the pathogenicity of VUS, but provides surrogate parameters for the monitoring of disease progression and therapeutic interventions. Lipids are essential organic compounds in living organisms, serving as building blocks for cellular membranes, energy storage and signaling molecules. Among other disorders, an imbalance in lipid homeostasis can lead to chronic inflammation, vascular dysfunction and neurodegenerative diseases. Therefore, analyzing lipids in biological samples provides great insight into the underlying functional role of lipids in healthy and disease statuses. The method of choice for lipid analysis and/or huge assemblies of lipids (=lipidome) is mass spectrometry due to its high sensitivity and specificity. Due to the inherent chemical complexity of the lipidome and the consequent challenges associated with analyzing it, progress in the field of lipidomics has lagged behind other omics disciplines. However, compared to the previous decade, the output of publications on lipidomics has increased more than 17-fold within the last decade and has, therefore, become one of the fastest-growing research fields. Combining multiple omics approaches will provide a unique and efficient tool for determining pathogenicity of VUS at the functional level, and thereby identifying rare, as well as novel, genetic disorders by molecular techniques and biochemical analyses.

Metabolomics analysis of antiquitin deficiency in cultured human cells and plasma: Relevance to pyridoxine-dependent epilepsy.

Deficiency of antiquitin (α-aminoadipic semialdehyde dehydrogenase), an enzyme involved in lysine degradation and encoded by ALDH7A1, is the major cause of vitamin B6 -dependent epilepsy (PDE-ALDH7A1). Despite seizure control with high dose pyridoxine (PN), developmental delay still occurs in approximately 70% of patients. We aimed to investigate metabolic perturbations due to possible previously unidentified roles of antiquitin, which may contribute to developmental delay, as well as metabolic effects of high dose pyridoxine supplementation reflecting the high doses used for seizure control in patients with PDE-ALDH7A1. Untargeted metabolomics by high resolution mass spectrometry (HRMS) was used to analyze plasma of patients with PDE-ALDH7A1 and two independently generated lines of cultured ReNcell CX human neuronal progenitor cells (NPCs) with CRISPR/Cas mediated antiquitin deficiency. Accumulation of lysine pathway metabolites in antiquitin-deficient NPCs and western-blot analysis confirmed knockdown of ALDH7A1. Metabolomics analysis of antiquitin-deficient NPCs in conditions of lysine restriction and PN supplementation identified changes in metabolites related to the transmethylation and transsulfuration pathways and osmolytes, indicating a possible unrecognized role of antiquitin outside the lysine degradation pathway. Analysis of plasma samples of PN treated patients with PDE-ALDH7A1 and antiquitin-deficient NPCs cultured in conditions comparable to the patient plasma samples demonstrated perturbation of metabolites of the gamma-glutamyl cycle, suggesting potential oxidative stress-related effects in PN-treated patients with PDE-ALDH7A1. We postulate that a model of human NPCs with CRISPR/Cas mediated antiquitin deficiency is well suited to characterize previously unreported roles of antiquitin, relevant to this most prevalent form of pyridoxine-dependent epilepsy.

Changes in the Cerebrospinal Fluid and Plasma Lipidome in Patients with Rett Syndrome.

Rett syndrome (RTT) is defined as a rare disease caused by mutations of the methyl-CpG binding protein 2 (MECP2). It is one of the most common causes of genetic mental retardation in girls, characterized by normal early psychomotor development, followed by severe neurologic regression. Hitherto, RTT lacks a specific biomarker, but altered lipid homeostasis has been found in RTT model mice as well as in RTT patients. We performed LC-MS/MS lipidomics analysis to investigate the cerebrospinal fluid (CSF) and plasma composition of patients with RTT for biochemical variations compared to healthy controls. In all seven RTT patients, we found decreased CSF cholesterol levels compared to age-matched controls (n = 13), whereas plasma cholesterol levels were within the normal range in all 13 RTT patients compared to 18 controls. Levels of phospholipid (PL) and sphingomyelin (SM) species were decreased in CSF of RTT patients, whereas the lipidomics profile of plasma samples was unaltered in RTT patients compared to healthy controls. This study shows that the CSF lipidomics profile is altered in RTT, which is the basis for future (functional) studies to validate selected lipid species as CSF biomarkers for RTT.

Asymmetric opening of the homopentameric 5-HT3A serotonin receptor in lipid bilayers.

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, 'coupled' state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.

A Metabolomics Workflow for Analyzing Complex Biological Samples Using a Combined Method of Untargeted and Target-List Based Approaches.

In the highly dynamic field of metabolomics, we have developed a method for the analysis of hydrophilic metabolites in various biological samples. Therefore, we used hydrophilic interaction chromatography (HILIC) for separation, combined with a high-resolution mass spectrometer (MS) with the aim of separating and analyzing a wide range of compounds. We used 41 reference standards with different chemical properties to develop an optimal chromatographic separation. MS analysis was performed with a set of pooled biological samples human cerebrospinal fluid (CSF), and human plasma. The raw data was processed in a first step with Compound Discoverer 3.1 (CD), a software tool for untargeted metabolomics with the aim to create a list of unknown compounds. In a second step, we combined the results obtained with our internally analyzed reference standard list to process the data along with the Lipid Data Analyzer 2.6 (LDA), a software tool for a targeted approach. In order to demonstrate the advantages of this combined target-list based and untargeted approach, we not only compared the relative standard deviation (%RSD) of the technical replicas of pooled plasma samples (n = 5) and pooled CSF samples (n = 3) with the results from CD, but also with XCMS Online, a well-known software tool for untargeted metabolomics studies. As a result of this study we could demonstrate with our HILIC-MS method that all standards could be either separated by chromatography, including isobaric leucine and isoleucine or with MS by different mass. We also showed that this combined approach benefits from improved precision compared to well-known metabolomics software tools such as CD and XCMS online. Within the pooled plasma samples processed by LDA 68% of the detected compounds had a %RSD of less than 25%, compared to CD and XCMS online (57% and 55%). The improvements of precision in the pooled CSF samples were even more pronounced, 83% had a %RSD of less than 25% compared to CD and XCMS online (28% and 8% compounds detected). Particularly for low concentration samples, this method showed a more precise peak area integration with its 3D algorithm and with the benefits of the LDAs graphical user interface for fast and easy manual curation of peak integration. The here-described method has the advantage that manual curation for larger batch measurements remains minimal due to the target list containing the information obtained by an untargeted approach.

Amyloid-beta impairs insulin signaling by accelerating autophagy-lysosomal degradation of LRP-1 and IR-ß in blood-brain barrier endothelial cells in vitro and in 3XTg-AD mice.

Aberrant insulin signaling constitutes an early change in Alzheimer's disease (AD). Insulin receptors (IR) and low-density lipoprotein receptor-related protein-1 (LRP-1) are expressed in brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). There, insulin may regulate the function of LRP-1 in Aß clearance from the brain. Changes in IR-ß and LRP-1 and insulin signaling at the BBB in AD are not well understood. Herein, we identified a reduction in cerebral and cerebrovascular IR-ß levels in 9-month-old male and female 3XTg-AD (PS1M146V, APPSwe, and tauP301L) as compared to NTg mice, which is important in insulin mediated signaling responses. Reduced cerebral IR-ß levels corresponded to impaired insulin signaling and LRP-1 levels in brain. Reduced cerebral and cerebrovascular IR-ß and LRP-1 levels in 3XTg-AD mice correlated with elevated levels of autophagy marker LC3B. In both genotypes, high-fat diet (HFD) feeding decreased cerebral and hepatic LRP-1 expression and elevated cerebral Aß burden without affecting cerebrovascular LRP-1 and IR-ß levels. In vitro studies using primary porcine (p)BCEC revealed that Aß peptides 1-40 or 1-42 (240 nM) reduced cellular levels and interaction of LRP-1 and IR-ß thereby perturbing insulin-mediated signaling. Further mechanistic investigation revealed that Aß treatment accelerated the autophagy-lysosomal degradation of IR-ß and LRP-1 in pBCEC. LRP-1 silencing in pBCEC decreased IR-ß levels through post-translational pathways further deteriorating insulin-mediated responses at the BBB. Our findings indicate that LRP-1 proves important for insulin signaling at the BBB. Cerebral Aß burden in AD may accelerate LRP-1 and IR-ß degradation in BCEC thereby contributing to impaired cerebral and cerebromicrovascular insulin effects.

Astaxanthin exerts protective effects similar to bexarotene in Alzheimer's disease by modulating amyloid-beta and cholesterol homeostasis in blood-brain barrier endothelial cells.

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-ß peptides (Aß) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aß production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aß oligomers and ~80 kDa intracellular 6E10-reactive APP/Aß species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aß clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aß species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aß oligomers in mBCEC and Aß species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aß from cerebrovascular endothelial cells.

Gestational diabetes mellitus modulates cholesterol homeostasis in human fetoplacental endothelium.

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H2DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography-mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [3H]-cholesterol labeled HPEC and [14C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7ß-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL3 (78 ±â€¯17%, 40 ±â€¯9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-ß clearance in blood-brain barrier endothelial cells.

Amyloid-ß peptides (Aß) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aß metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aß. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aß metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aß oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aß clearance across the pBCEC monolayer. Treatment of pBCEC with Aß(1-40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aß oligomers and reduced Aß uptake and cell-associated Aß oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aß processing and clearance at the BBB.