Biology subjects

Hornemann, T.

Publications and source records attributed to Hornemann, T..

13 recordsLinked to original sources

bioRxiv2026

CYP4F2-mediated ω-hydroxylation of 1-deoxysphingolipids reveals a new hepatic detoxification pathway

1-deoxysphingolipids (1-deoxySLs) are atypical, cytotoxic sphingolipids (SL) formed by the serine palmitoyltransferase through the alternative use of L-Alanine over its canonical substrate L-Serine. Elevated plasma levels of 1-deoxySLs have been implicated in metabolic and neurodegenerative diseases. Due to the missing C1 hydroxyl group, 1-deoxySLs cannot be converted into complex sphingolipids nor degraded via the canonical SL catabolic pathways. However, previous reports suggested a cytochrome P450 mediated {omega}-hydroxylation of 1-deoxySLs as a potential detoxification mechanism although the exacts downstream metabolism of these lipids remained unclear. We combined genome-wide association analysis with targeted lipid analysis to identify genes involved in 1-deoxySL metabolism. Functional validation was performed in cell culture models, enzyme assays, and through quantitative high-resolution mass spectrometry using isotope labelled synthetic standards.We identified a strong association between the CYP4F2 rs2108622 variant and plasma 1-deoxySL, implicating CYP4F2 is involved in 1-deoxySL metabolism. We demonstrated that CYP4F2 catalyzes the {omega}-hydroxylation of 1-deoxysphinganine, forming a previously uncharacterized hydroxylated sphingoid base. In liver cells, this metabolite was further metabolized via three distinct pathways: one forming the N-acyl, a second involving omega acylation and third resulting in omega carboxylation. All reactions generated a new spectrum of 1-deoxysphingolipids that are based on {omega}-hydroxylated 1-deoxySA as a precursor. The metabolic steps were confirmed by structural validation using synthetically prepared external standards. Importantly, {omega}-hydroxylation significantly attenuated the acute cytotoxicity of 1-deoxySLs in liver cells, indicating that this modification is the initiating step of a multi-branched metabolic clearance pathway. This study identifies CYP4F2 as a key enzyme initiating the hepatic clearance of atypical 1-deoxySLs, mitigating their cellular toxicity and revealing multiple downstream metabolic fates. Our findings highlight a previously unrecognized clearance mechanism for atypical sphingolipids with relevance to metabolic disease.

biochemistry↗

bioRxiv2026

Sphingolipid remodelling in SPT-related neuropathies

Sphingolipid homeostasis is critical for neuronal structural and functional integrity in the central and peripheral nervous systems. The rate-limiting enzyme of this pathway, serine-palmitoyltransferase (SPT), establishes the metabolic entry point into sphingolipid biosynthesis. Mutations in the SPT subunits, SPTLC1 and SPTLC2 lead to contrasting disease phenotypes in patients, including amyotrophic lateral sclerosis (ALS) and hereditary sensory neuropathy (HSAN1). A third mixed sensory-motor phenotype is attributed to distinct mutation sets in SPTLC1 and SPTLC2. However, a direct comparison of the metabolic consequences of mutations spanning these disease conditions has not been performed. Here, we demonstrate that SPTLC1- and SPTLC2-ALS variants contribute to enhanced sphingolipid flux while the ceramide-mediated homeostatic control is impaired. In contrast, HSAN1-associated variants display altered substrate selectivity, shifting flux towards non-canonical 1-deoxysphingolipid (1-deoxySL) production but decreasing canonical synthesis. The variant associated with a mixed sensory-motor phenotype exhibit a third metabolic state with elevated 1-deoxySL formation and, in contrast to HSAN1-variants, increased canonical sphingolipid synthesis. Sphingolipid profiling reveals that ALS variants are characterized by preferential accumulation of dihydro- and intermediate chain sphingolipid species. Notably, the separation of lipid species between ALS and HSAN1 is robust, with canonical sphingolipids enriched in ALS variants, while long-chain 1-deoxySL dominate in HSAN1. The SPT-variants associated with mixed sensory and motor symptoms are associated with elevated levels of both types. The data support the view that segregated shifts in sphingolipid flux underlie divergence of clinical phenotypes in SPT-variants and offer guidance for therapeutic interventions. Importantly, therapeutic strategies must account for these metabolic configurations, as L-serine supplementation may benefit HSAN1 but exacerbate pathology in ALS and sensory-motor disease conditions.

neuroscience↗

bioRxiv2025

Dyslipidemic SPTLC3 Integrates Bile Acid-FXR Signaling with Sphingolipid Remodeling in MASLD

Molecular mechanisms driving metabolic disease pathogenesis remain poorly understood. Genetic and functional studies implicate SPTLC3 with dyslipidemia. SPTLC3 synthesizes atypical long-chain bases (LCB) precursors for sphingolipid production. We demonstrate significant SPTLC3 expression in human liver. ORMDL1-3 regulate SPTLC3 post-translationally in hepatic and non-hepatic cells. Independently, farnesoid X receptor (FXR) represses hepatic SPTLC3 transcription via a negative promoter element. In mice, high-fat diet (HFD) induced whereas bile acids normalized hepatic SPTLC3 transcription. SPTLC3 derived LCBs in plasma originate from the liver and are elevated in HFD-fed mice and in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Cross-species comparison revealed marked differences in LCB composition between mice and humans. Notably, omega-3-methylsphingosine (meC18SO) was significantly associated with MASLD in humans but undetectable in mice. In Huh7 cells, meC18SO enhanced complex II and IV activity, oxygen consumption, and mitochondrial ROS content. FXR-SPTLC3 axis and presented findings have potential implications for future translational research.

biochemistry↗

bioRxiv2025

Very long chain fatty acids drive 1-deoxy-Sphingolipid toxicity

1-deoxy-sphingolipids (1-deoxySLs) are atypical sphingolipids synthesized by the serine palmitoyltransferase (SPT) when L-alanine is used instead of its canonical substrate L-serine. Increased 1-deoxySLs are associated with sensory neuropathies such as Hereditary Sensory and Autonomic Neuropathy type 1 (HSAN1) and diabetic polyneuropathy (DPN). Despite their known cellular, mitochondrial, and neurotoxic effects, the mechanisms underlying their toxicity remain poorly understood. Using a CRISPR interference (CRISPRi) screening approach, we identified CERS2, ELOVL1, ACACA, HSD17B12, and PTPLB as key mediators of 1-deoxySL-induced toxicity. All genes are integral to the biosynthesis of very long-chain (VLC) fatty acids and VLC-ceramides. We validated these findings through genetic knockdown experiments, cytotoxicity assays, and stable isotope-resolved lipidomics via LC-MS/MS. Pharmacological inhibition of ELOVL1 using a preclinical tested compound alleviated the cellular, mitochondrial, and neuronal toxicity induced by 1-deoxySLs. Supplementation experiments combining 1-deoxySLs with various VLC fatty acids revealed that 1-deoxyDHceramide conjugated to nervonic acid (m18:0/24:1) is the principal toxic specie. Further mechanistic studies showed that m18:0/24:1 induces apoptosis through the mitochondrial permeability transition pore (mPTP) formation. Inhibition of BAX or blocking mPTP formation with cyclosporin A effectively prevented toxicity. In conclusion, our findings demonstrate that 1-deoxyDHCeramides conjugated to nervonic acid are the primary mediators of 1-deoxySL toxicity, acting through mitochondrial dysfunction and BAX-dependent apoptosis.

biochemistry↗

bioRxiv2025

Metabolic Origins of Neurotoxic 1-deoxySphingolipids in Type 2 Diabetes

Type 2 diabetes (T2D) and diabetic peripheral neuropathy (DPN) are associated with disruptions in sphingolipid (SL) metabolism, including an increased formation of neurotoxic 1-deoxysphingolipids (1-deoxySL). Here we report data from an untargeted proteomics, lipidomics and metabolomics profiling in plasma and skin samples of a carefully characterized T2D cohort and age-matched healthy controls. We investigated the association between plasma and skin amino acids and the sphingolipidome in blood and skin of T2D patients and several diabetic rodent models. We developed a hypothesis on how changes in the metabolism of the two amino acids relates to SL formation and DPN. To test this hypothesis and identify key enzymes responsible for the 1-deoxySL formation we developed stable isotope based SL flux assays, using UC13Glucose, 15NGlutamine, D4-Palmitic acid, D4-Alanine and D3N15-Serine as tracers. In combination with genetic interference approaches, we identified pathways that are responsible for shifting between the formation of 1-deoxySL and canonical SL in T2D. Furthermore, we verified these findings in vivo in several rodent models. This study links disturbances in amino acids, lipids, and protein homeostasis to DPN in T2D.

neuroscience↗

bioRxiv2025

Disrupted Sphingosine-1-Phosphate Homeostasis Drives Nephrotoxicity in Sphingosine-1-Phosphate Lyase Insufficiency Syndrome (SPLIS)

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS), also known as nephrotic syndrome type 14 (NPHS14), is an autosomal recessive disorder characterized by renal, neurological, dermatological, endocrine, and immunological symptoms. This condition is caused by loss-of-function mutations in the SGPL1 gene, which encodes sphingosine-1-phosphate lyase (SGPL1p/SPL), the enzyme responsible for the terminal degradation of sphingosine-1-phosphate (S1P) in sphingolipid catabolism. We investigated a novel case of SPLIS associated with a recently reported SGPL1 mutation (c.1084T>A; p.Ser362Thr). Using stable isotope flux analyses, we demonstrated in patient-derived fibroblasts and HEK293T SGPL1 knockout models that SGPL1p deficiency does not consistently result in pathological S1P accumulation. Instead, SPL-deficient cells are able to maintain steady-state S1P levels through two compensatory mechanisms: O_LIRegulation of de novo sphingolipid synthesis via the ORMDL-ceramide axis. C_LIO_LIIncreased conversion of excess ceramides into glycosphingolipids. C_LI However, when steady-state conditions are disrupted--either by external sphingolipid supplementation or by impairing homeostatic control--a pathological increase in intracellular S1P occurs in SPL-deficient cells. In vivo, Sgpl1-/-mice exhibited significant urinary excretion of S1P and marked S1P enrichment in the kidneys. This pathological accumulation of S1P dysregulates cytoskeletal homeostasis, impairing renal epithelial formation. Based on these findings, we hypothesize that the reabsorption of urinary S1P contributes to toxic renal accumulation, providing an explanation for the nephrotoxicity observed in SPLIS and its association with nephrotic syndrome. Importantly, we found that the cytoskeletal disruptions could be mitigated by inhibiting the Rho-ROCK signaling pathway using the clinically approved inhibitor Fasudil. These findings illuminate the pathophysiological basis of SPLIS nephrotoxicity and propose a promising pharmacological intervention strategy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/634100v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@e70e68org.highwire.dtl.DTLVardef@1630198org.highwire.dtl.DTLVardef@fde277org.highwire.dtl.DTLVardef@1f2b8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Chromatin Remodeling by the Histone Methyltransferase SETD2 Drives Lipotoxic Injury in Cardiometabolic Heart Failure with Preserved Ejection Fraction

BackgroundCardiometabolic heart failure with preserved ejection fraction (cHFpEF) is highly prevalent and associates with a poor outcome. Pathological gene expression in heart failure is accompanied by changes in active histone marks without major alterations in DNA methylation. Histone 3 trimethylation at lysine 36 (H3k36me3) - a chromatin signature induced by the histone methyltransferase SETD2 - strongly correlates with changes in gene expression in human failing hearts; however, its role is poorly understood. Here we investigate the role of SETD2 in cHFpEF. MethodsMice with cardiomyocyte-specific deletion of SETD2 (c-SETD2-/-) were generated and subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Cardiac function and exercise tolerance were assessed by echocardiography and Treadmill exhaustion test. Chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD2/H3k36me3 enrichment on gene promoters. SETD2 gain- and loss-of-function experiments were performed in cultured cardiomyocytes (CMs) exposed to palmitic acid (PA). SETD2 expression was also investigated in left ventricular (LV) myocardial specimens from patients with cHFpEF and control donors. ResultsSETD2 was upregulated in cHFpEF mouse hearts and its chromatin mark H3k36me3 was enriched on the promoter of sterol regulatory element-binding transcription factor 1 (SREBP1) gene. SETD2 activation in cHFpEF led to SREBP1 upregulation, triglyceride accumulation and lipotoxic damage. Of note, cardiomyocyte-specific deletion of SETD2 in mice prevented HFpEF-related hypertrophy, diastolic dysfunction and lung congestion while improving exercise tolerance. SETD2 deletion blunted H3K36me3 enrichment on SREBP1 promoter thus leading to a marked rewiring of the cardiac lipidome and restoration of autophagic flux. SETD2 depletion in PA-treated CMs prevented SREBP1 upregulation, whereas SETD2 overexpression recapitulated lipotoxic damage. Finally, SETD2 was upregulated in LV specimens from cHFpEF patients and its pharmacological inhibition by EZM0414 attenuated CM stiffness. ConclusionsTherapeutic modulation of SETD2/H3k36me3 axis might prevent lipotoxic injury and cardiac dysfunction in cHFpEF.

physiology↗

bioRxiv2022

ROS induction as a strategy to target persister cancer cells with low metabolic activity in NRAS mutated melanoma

Metabolic reprogramming is an emerging hallmark of resistance to cancer therapy but may generate vulnerabilities that can be targeted with small molecules. Multi-omics analysis revealed that NRAS-mutated melanoma cells with a mesenchymal transcriptional profile adopt a quiescent metabolic program to resist cellular stress response induced by MEK-inhibitor resistance. However, as a result of elevated baseline ROS levels, these cells become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intra-tumor heterogeneity requires the combination of a ROS-inducer and a MEK-inhibitor to target both tumor growth and metastasis. By ex vivo pharmacoscopy of 62 human metastatic melanomas, we found that MEK-inhibitor resistant tumors significantly benefitted from the combination therapy. Finally, we profiled 486 cancer cell lines and revealed that oxidative stress responses and translational suppression are biomarkers of ROS-inducer sensitivity, independent of cancer indication. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS-inducers in melanoma and other cancers. Statement of SignificanceTargeted-therapy resistance in cancer arises from genetic selection and both transcriptional and metabolic adaptation. We show that metabolic reprogramming sensitizes resistant cells to ROS-induction in combination with pathway inhibitors. Predictive biomarkers of metabolic sensitivity to ROS-inducing agents were identified in many cancer entities, highlighting the generalizability of this treatment approach. Graphical summary O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cancer biology↗

bioRxiv2022

SPTLC1 variants associated with childhood onset amyotrophic lateral sclerosis produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Mutations in the SPTLC1 subunit of serine-palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased sphingolipid synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause the peripheral sensory neuropathy HSAN1 due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

biochemistry↗

bioRxiv2021

The Bardet-Biedl protein Bbs1 controls photoreceptor outer segment protein and lipid composition

Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a new bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on isolated OSs show that Bbs1 is required for BBSome-entry into OSs and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a new role for Bbs1/BBSome in OS lipid homeostasis and suggest a new pathomechanism underlying retinal degeneration in BBS.

molecular biology↗

bioRxiv2021

Discovery and mechanism of action of small molecule inhibitors of ceramidases

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes such as energy utilisation and cell proliferation. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to ultimately produce pro-proliferative sphingosine-1-phosphate. Human ceramidases can be soluble proteins (acid and neutral ceramidase) or integral membrane proteins (alkaline ceramidases). Increasing ceramide levels to increase apoptosis has shown efficacy as a cancer treatment using small molecules inhibiting a soluble ceramidase. Due to the transmembrane nature of alkaline ceramidases, no specific small molecule inhibitors have been reported. Here, we report novel fluorescent substrates (FRETceramides) of ceramidases that can be used to monitor enzyme activity in real-time. We use FRETceramides to discover the first drug-like inhibitors of alkaline ceramidase 3 (ACER3) which are active in cell-based assays. Biophysical characterization of enzyme:inhibitor interactions reveal a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules. Table of contents summaryUse of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug-like small molecule inhibitors for alkaline ceramidase 3, an intra-membrane enzyme involved in sphingolipid metabolism in health and disease.

biochemistry↗

bioRxiv2020

A global lipid map defines a network essential for Zika virus replication

Zika virus (ZIKV), an arbovirus of global concern, remodels intracellular membranes to form replication sites. How ZIKV dysregulates lipid networks to allow this, and consequences for disease, is poorly understood. Here, we performed comprehensive lipidomics to create a lipid network map during ZIKV infection. We found that ZIKV significantly alters host lipid composition, with the most striking changes seen within subclasses of sphingolipids. Ectopic expression of ZIKV NS4B protein resulted in similar changes, demonstrating a role for NS4B in modulating sphingolipid pathways. Disruption of sphingolipid biosynthesis in various cell types, including human neural progenitor cells, blocked ZIKV infection. Additionally, the sphingolipid ceramide redistributes to ZIKV replication sites and increasing ceramide levels by multiple pathways sensitizes cells to ZIKV infection. Thus, we identify a sphingolipid metabolic network with a critical role in ZIKV replication and show that ceramide flux is a key mediator of ZIKV infection.

microbiology↗

bioRxiv2018

Aberrant DEGS1 sphingolipid metabolism impairs central and peripheral nervous system function in humans

Sphingolipids including ceramides are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids are associated with various neurological pathologies, however, the entire spectrum of disorders affecting sphingolipid metabolism remains elusive. By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous system, we identified a homozygous variant p.(Ala280Val) in DEGS1, encoding an enzyme of the ceramide synthesis pathway. The blood sphingolipid profile and patient-derived fibroblasts both showed a significant shift from the unsaturated to the dihydro-forms of sphingolipids. Moreover, an atypical and potentially toxic sphingolipid metabolite is formed as consequence of the altered synthesis pathway. The changes in the sphingolipid profile were recapitulated in a CRISPR/Cas-based DEGS1 knockout HAP1-cell model and by chemical inhibition of DEGS1, suggesting a loss of DEGS1 function in the disease. DEGS1 insufficiency is thus a novel cause for a multisystem neurological disorder. A sphingolipid-rich diet may correct the metabolic profile and improve the clinical outcome of affected individuals and suggests that this heritable condition might be treatable.\n\nAbbreviations

genetics↗