ceramide species  (Croda International Plc)

Journal: The Journal of Biological Chemistry

Article Title: Acid ceramidase ASAH1 is a key regulator of epidermal ceramide levels and composition

doi: 10.1016/j.jbc.2026.111178

Figure Lengend Snippet: Ceramide class–dependent accumulation in ASAH1 KO keratinocytes. A and B , lipids were extracted from control ( A and B ), ASAH1 KO ( A – C ), and ACER1 KO ( A ) keratinocytes under undifferentiated conditions ( A ) and after 14 days of differentiation ( A – C ), and the indicated classes of ceramides ( A and B ), hexosylceramides ( C ), and sphingomyelins ( C ) were quantified via LC–MS/MS. Values presented are mean + SD of total quantities of the indicated lipid classes ( A and C ) and individual ceramide species categorized by FA moiety ( B ) (n = 3; ∗ p < 0.05; ∗∗ p < 0.01; Dunnett’s test versus control in A , Welch’s t test in B ). Diff, differentiated; HexCer, hexosylceramide; Undiff, undifferentiated.

Article Snippet: A mixture of 60 μl of 5 mg/ml 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (16:0/18:1 phosphatidylcholine; Avanti Research), dissolved in chloroform/methanol (1:1, v/v), and 40 μl of each ceramide species at 100 μM— N -palmitoyl- d - erythro -sphingosine (C16:0 NS; Avanti Research), N -lignoceroyl- d - erythro -sphingosine (C24:0 NS; Avanti Research), N -nervonoyl- d - erythro -sphingosine (C24:1 NS; Avanti Research), N -lignoceroyl- d - erythro -dihydrosphingosine (C24:0 NdS; Avanti Research), N -lignoceroyl- d - ribo -phytosphingosine (C24:0 NP; Avanti Research), N -lignoceroyl-6-( R )-hydroxysphingosine (C24:0 NH; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - erythro -sphingosine (C24:0 AS; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - ribo -phytosphingosine (C24:0 AP; Avanti Research), N -(30-linoleoyloxy-triacontanoyl)- d - erythro -sphingosine (C30:0 EOS; Cayman Chemical), and N -ω-hydroxytriacontanoyl- d - erythro -sphingosine (C30:0 OS; Cayman Chemical)—was combined and dried.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Acid ceramidase ASAH1 is a key regulator of epidermal ceramide levels and composition

doi: 10.1016/j.jbc.2026.111178

Figure Lengend Snippet: Ceramide class–specific activity of ASAH1. HEK 293T cells were transfected with either the pCE-puro 3× FLAG-4 vector or the pCE-puro ASAH1-3× FLAG plasmid. Culture supernatant was collected 48 h after transfection as the enzyme source. A , proteins were concentrated via precipitation with 5% trichloroacetic acid, followed by SDS-PAGE and immunoblotting using an anti-FLAG antibody. B , the indicated ceramide species (final concentration, 5 μM; 600 pmol) were incorporated into liposomes and incubated with the culture supernatant from the pCE-puro ASAH1-3× FLAG plasmid-transfected cells (containing 145 ng ASAH1-3× FLAG) or with supernatant from vector-transfected cells as a negative control. After incubation, lipids were extracted, and the resulting long-chain bases (ceramide degradation products) were quantified via LC–MS/MS. Values presented are mean + SD of the quantity of long-chain bases produced per minute by 1 μg of ASAH1-3× FLAG, expressed in picomoles (n = 3; ∗ p < 0.05; ∗∗ p < 0.01; Welch’s t test). HEK, human embryonic kidney cell line.

Article Snippet: A mixture of 60 μl of 5 mg/ml 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (16:0/18:1 phosphatidylcholine; Avanti Research), dissolved in chloroform/methanol (1:1, v/v), and 40 μl of each ceramide species at 100 μM— N -palmitoyl- d - erythro -sphingosine (C16:0 NS; Avanti Research), N -lignoceroyl- d - erythro -sphingosine (C24:0 NS; Avanti Research), N -nervonoyl- d - erythro -sphingosine (C24:1 NS; Avanti Research), N -lignoceroyl- d - erythro -dihydrosphingosine (C24:0 NdS; Avanti Research), N -lignoceroyl- d - ribo -phytosphingosine (C24:0 NP; Avanti Research), N -lignoceroyl-6-( R )-hydroxysphingosine (C24:0 NH; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - erythro -sphingosine (C24:0 AS; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - ribo -phytosphingosine (C24:0 AP; Avanti Research), N -(30-linoleoyloxy-triacontanoyl)- d - erythro -sphingosine (C30:0 EOS; Cayman Chemical), and N -ω-hydroxytriacontanoyl- d - erythro -sphingosine (C30:0 OS; Cayman Chemical)—was combined and dried.

Techniques: Activity Assay, Transfection, Plasmid Preparation, SDS Page, Western Blot, Concentration Assay, Liposomes, Incubation, Negative Control, Liquid Chromatography with Mass Spectroscopy, Produced

Journal: The Journal of Biological Chemistry

Article Title: Acid ceramidase ASAH1 is a key regulator of epidermal ceramide levels and composition

doi: 10.1016/j.jbc.2026.111178

Figure Lengend Snippet: Enzyme kinetic parameters of ASAH1. HEK 293T cells were transfected with either the pCE-puro 3× FLAG-4 vector or the pCE-puro ASAH1-3× FLAG plasmid. Culture supernatant was collected 48 h after transfection as the enzyme source. The indicated ceramide species with different classes—C24:0 NS ( A ), C24:0 NP ( B ), C24:0 NH ( C ), and C24:0 AS ( D )—were incorporated into liposomes at final concentrations of 0.4, 0.8, 1.6, and 3.2 μM and incubated with the culture supernatant at 37 °C for 2 h. After incubation, lipids were extracted, and the resulting long-chain bases (ceramide degradation products) were quantified via LC–MS/MS. The data obtained were used to generate Lineweaver–Burk plots by plotting the inverse of reaction velocity (1/ V ) against the inverse of substrate concentration (1/[S]). Values presented are mean ± SD (n = 3). Values of the calculated kinetic parameters ( K M , V max , and k cat / K M ) are also shown. HEK, human embryonic kidney cell line.

Article Snippet: A mixture of 60 μl of 5 mg/ml 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (16:0/18:1 phosphatidylcholine; Avanti Research), dissolved in chloroform/methanol (1:1, v/v), and 40 μl of each ceramide species at 100 μM— N -palmitoyl- d - erythro -sphingosine (C16:0 NS; Avanti Research), N -lignoceroyl- d - erythro -sphingosine (C24:0 NS; Avanti Research), N -nervonoyl- d - erythro -sphingosine (C24:1 NS; Avanti Research), N -lignoceroyl- d - erythro -dihydrosphingosine (C24:0 NdS; Avanti Research), N -lignoceroyl- d - ribo -phytosphingosine (C24:0 NP; Avanti Research), N -lignoceroyl-6-( R )-hydroxysphingosine (C24:0 NH; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - erythro -sphingosine (C24:0 AS; Avanti Research), N -(2'-( R )-hydroxylignoceroyl)- d - ribo -phytosphingosine (C24:0 AP; Avanti Research), N -(30-linoleoyloxy-triacontanoyl)- d - erythro -sphingosine (C30:0 EOS; Cayman Chemical), and N -ω-hydroxytriacontanoyl- d - erythro -sphingosine (C30:0 OS; Cayman Chemical)—was combined and dried.

Techniques: Transfection, Plasmid Preparation, Liposomes, Incubation, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: NPJ Parkinson's Disease

Article Title: Inhibition of de novo ceramide synthesis mitigates alpha-synuclein pathology in a Parkinson’s disease mouse model

doi: 10.1038/s41531-026-01263-5

Figure Lengend Snippet: a Quantification of ceramide species in postmortem midbrain tissues from individuals with Lewy body dimentia (LBD; n = 6) and age-matched neurologically healthy controls ( n = 6) using targeted UHPLC-MS/MS. Bar graph shows ceramide species significantly increased in LBD tissue compared to control tissue (fold-change > 1.5; FDR-adjusted p < 0.05). Long-chain acyl ceramides (e.g., C16:0, C18:0, C22:0, C27:0) were among the most elevated in LBD brains. b UMAP visualization of single-nucleus RNA-seq data ( GSE243639 ) from midbrain samples of PD patients and controls. Unsupervised clustering identified multiple neuronal and non-neuronal subpopulations. Cluster identity is color-coded. c Dot plot showing the expression of canonical neuronal markers across neuronal subtypes. Dopaminergic neuron identity was assigned to cluster_0 based on high expression of TH and SLC6A3 . d Heatmap displaying Z-score standardized expression of ceramide biosynthesis and metabolism genes in dopaminergic neurons from Parkinson’s disease (PD) patients and healthy controls. Each gene’s expression was standardized across groups to highlight relative differences. Visual inspection suggests higher expression of CERS5, CERS6, DEGS1, and GBA in PD. e Violin plots show module scores for ceramide biosynthesis genes across 22 transcriptionally defined cell types identified in bulk RNA-seq dataset GSE156776 . comparing Con (gray) and IPD (yellow) groups. Scores were calculated using Seurat’s AddModuleScore with genes involved in ceramide synthesis. Statistical significance was assessed using the Wilcoxon rank-sum test (ns not significant, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). f Gene ontology (GO) enrichment analysis of ceramide biosynthesis–associated genes. Bar graph shows significantly enriched GO biological processes related to ceramide metabolism (FDR < 0.05). Data presented as gene count per GO term and adjusted p -value.

Article Snippet: To assess the effects of exogenous ceramide species on α-synuclein pathology, Day 50 midbrain organoids were treated with either C16 Ceramide (d18:1/16:0; Avanti Polar Lipids, Cat# 10681) or C24:1 Ceramide (d18:1/24:1(15Z); Avanti Polar Lipids, Cat# 62530) at a final concentration of 10 μM for 48 h. Ceramide stock solutions were prepared in ethanol and diluted in organoid maintenance medium immediately prior to administration.

Techniques: Tandem Mass Spectroscopy, Control, RNA Sequencing, Expressing, Process/Product Development

Journal: NPJ Parkinson's Disease

Article Title: Inhibition of de novo ceramide synthesis mitigates alpha-synuclein pathology in a Parkinson’s disease mouse model

doi: 10.1038/s41531-026-01263-5

Figure Lengend Snippet: a Schematic overview of the treatment regimen in A53T α-synuclein transgenic mice (M83 line). Myriocin (0.4 mg/kg, i.p.) or vehicle was administered three times per week starting at 5 months of age for either 5 months (E1) or 7 months (E2). Relative levels of ceramide species in ( b ) plasma and ( c ) midbrain tissue measured by ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS). b Quantification of ceramide species in plasma of wild-type (WT), vehicle-treated PD (PD-veh), and myriocin-treated PD (PD-myr) mice. Several long-chain ceramide species were significantly elevated in PD-veh mice and reduced by myriocin treatment. c Heatmap showing relative abundance of individual ceramide species in the midbrain of WT, PD-veh, and PD-myr mice. Ceramide levels were normalized to total lipid content. Myriocin reduced multiple ceramide species elevated in PD-veh mice. Asterisks indicate statistical significance compared with the PD-veh group (one-way ANOVA followed by Dunnett’s post hoc test). * p < 0.05, ** p < 0.01, n = 3 per group. d Representative locomotor traces from the open field test showing reduced center exploration and hypoactivity in PD-veh mice, partially restored by myriocin. e Behavioral quantification from open field testing. PD-veh mice exhibited reduced distance, time, and entries in the central zone as well as decreased total distance traveled, all significantly improved by myriocin treatment ( n = 3–5 mice per group). f Y-maze spontaneous alternation test. PD-veh mice showed impaired spatial working memory, as indicated by lower alternation percentage, significantly improved in the PD-myr group ( n = 3–5 mice per group). g Tyrosine hydroxylase (TH) immunohistochemistry of midbrain sections from PD-veh and PD-myr mice at 10 and 12 months of age. Myriocin preserved TH-positive neurons in the substantia nigra pars compacta and ventral tegmental area. Scale bars, 200 μm. h Immunofluorescence images showing DAPI (blue), TH (magenta), and phospho–α-synuclein (pS129, green) in the substantia nigra. Myriocin treatment reduced pathological pS129 α-synuclein immunoreactivity. Insets show magnified views of boxed regions. Scale bars, 30 μm. * p < 0.05; ** p < 0.01.

Article Snippet: To assess the effects of exogenous ceramide species on α-synuclein pathology, Day 50 midbrain organoids were treated with either C16 Ceramide (d18:1/16:0; Avanti Polar Lipids, Cat# 10681) or C24:1 Ceramide (d18:1/24:1(15Z); Avanti Polar Lipids, Cat# 62530) at a final concentration of 10 μM for 48 h. Ceramide stock solutions were prepared in ethanol and diluted in organoid maintenance medium immediately prior to administration.

Techniques: Transgenic Assay, Clinical Proteomics, High Performance Liquid Chromatography, Mass Spectrometry, Immunohistochemistry, Immunofluorescence

Journal: NPJ Parkinson's Disease

Article Title: Inhibition of de novo ceramide synthesis mitigates alpha-synuclein pathology in a Parkinson’s disease mouse model

doi: 10.1038/s41531-026-01263-5

Figure Lengend Snippet: a Bar plot showing the number of differentially expressed genes (DEGs; adjusted p < 0.05, |log₂FC | >0.58) across three brain regions (cortex, hippocampus, and midbrain) in pairwise comparisons: PD-veh vs. WT and PD-myr vs. PD-veh. The midbrain displayed the highest number of DEGs in response to myriocin treatment (PD-myr vs. PD-veh), indicating a region-specific transcriptional effect. b Volcano plots illustrating midbrain DEGs in PD-veh vs. WT (left) and PD-myr vs. PD-veh (right) comparisons. Red dots indicate upregulated genes and blue dots indicate downregulated genes (adjusted p < 0.05, |log₂FC | >0.58). Myriocin treatment reversed many inflammation-related gene changes observed in PD-veh mice. c Heatmap showing Z-score–normalized expression of genes downregulated in PD-veh mice but upregulated in both WT and PD-myr groups (rescue pattern 1). d Gene Ontology (GO) enrichment analysis of rescue-pattern genes in panel ( c ), indicating restored pathways related to synaptic transmission, learning, and cognition. e Heatmap showing Z-score–normalized expression of genes upregulated in PD-veh but decreased in WT and PD-myr groups (rescue pattern 2), many of which are associated with immune or inflammatory responses. f GO enrichment analysis of genes in ( e ), highlighting biological processes such as interferon signaling and innate immune response that were reversed by myriocin. g Gene Set Enrichment Analysis (GSEA) volcano plot showing gene sets significantly modulated by myriocin (PD-myr vs. PD-veh). Myriocin increased the expression of gene sets related to mitophagy, mitochondrial homeostasis, and neuronal signaling, while downregulating pathways associated with inflammation, apoptosis, and ceramide metabolism. All downstream transcriptomic analyses were performed on midbrain tissues ( n = 3 mice per group). DEGs were identified using DESeq2 with Benjamini–Hochberg FDR correction.

Article Snippet: To assess the effects of exogenous ceramide species on α-synuclein pathology, Day 50 midbrain organoids were treated with either C16 Ceramide (d18:1/16:0; Avanti Polar Lipids, Cat# 10681) or C24:1 Ceramide (d18:1/24:1(15Z); Avanti Polar Lipids, Cat# 62530) at a final concentration of 10 μM for 48 h. Ceramide stock solutions were prepared in ethanol and diluted in organoid maintenance medium immediately prior to administration.

Techniques: Expressing, Transmission Assay

Journal: NPJ Parkinson's Disease

Article Title: Inhibition of de novo ceramide synthesis mitigates alpha-synuclein pathology in a Parkinson’s disease mouse model

doi: 10.1038/s41531-026-01263-5

Figure Lengend Snippet: a Representative confocal images of mt-Keima–expressing patient-derived dopaminergic neurons following 48-h treatment with vehicle, 10 μM, or 50 μM myriocin. Cells were imaged at excitation wavelengths of 488 nm (neutral mitochondria, green) and 561 nm (acidified mitolysosomes, red). Merged and zoomed images (bottom) highlight increased red puncta upon myriocin treatment, indicating enhanced mitophagic flux. Scale bars, 50 μm. b Quantification of mitophagy levels expressed as the 561/488 nm excitation ratio. Data are presented as mean ± SEM from four independent experiments. * P < 0.05 by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. c Confocal immunofluorescence images of patient-derived neurons treated with vehicle or 10 μM myriocin for 48 h, stained for TOM20 (magenta), phospho-α-synuclein (pS129; green), and DAPI (blue). Myriocin restored mitochondrial network integrity. Scale bars, 50 μm. d Quantification of mitochondrial morphology in patient-derived neurons treated with vehicle or myriocin (10 μM or 50 μM) for 48 h. Morphological parameters, including mitochondrial area, perimeter, total branch length, and branch junctions per mitochondrion, were measured using Mitochondria Analyzer in ImageJ. Data represents SEM from n = 10 cells per condition. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons. e Immunofluorescence analysis of midbrain organoid sections from healthy controls, vehicle-treated PD lines, and myriocin-treated PD lines. Left: Co-staining for neuronal markers Tuj1 (green) and tyrosine hydroxylase (TH; red) revealed myriocin-mediated rescue of dopaminergic neuronal loss. Right: Immunostaining for phospho-α-synuclein (pS129; magenta) showed reduced aggregate levels in myriocin-treated organoids. Nuclei were stained with DAPI (blue). Scale bars, 100 μm. f Effects of exogenous ceramide supplementation on α-synuclein aggregation in midbrain organoids. Organoids derived from PD or healthy iPSC lines were treated with 10 μM of C16 and C24:1 ceramide mixture for 48 h and stained for pS129 (magenta) and DAPI (blue). Ceramide supplementation increased α-synuclein aggregation in PD-derived organoids. Scale bars, 100 μm.

Article Snippet: To assess the effects of exogenous ceramide species on α-synuclein pathology, Day 50 midbrain organoids were treated with either C16 Ceramide (d18:1/16:0; Avanti Polar Lipids, Cat# 10681) or C24:1 Ceramide (d18:1/24:1(15Z); Avanti Polar Lipids, Cat# 62530) at a final concentration of 10 μM for 48 h. Ceramide stock solutions were prepared in ethanol and diluted in organoid maintenance medium immediately prior to administration.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Staining, Immunostaining