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Transcriptomic analysis of HAP1 DEGS1 KO cells and their wildtype counterpart

ABSTRACT: Genetic variants in sphingolipid metabolizing enzymes are known to cause neurodegenerative and metabolic diseases by altering the cellular sphingolipid composition. Dysfunction of delta4-dihydroceramide (dhCer) desaturase (DEGS1) has recently been associated with a rare human monogenic neurological disease affecting sphingolipid (SL) homeostasis. Patients with pathogenic DEGS1 variants show elevated levels of different dhCer species and develop a hypomyelinating leukodystrophy in early childhood. To better understand the human pathophysiology and mechanistic complexity of this severe and progressive disease, we here utilized human DEGS1-knockout (KO) HAP1 cells as a genetic KO model to comprehensively investigate the effects of elevated dihydroceramide species on various intracellular processes, like autophagy, apoptosis, ROS formation and ER Stress by using flow cytometry, Western Blot and fluorescence microscopy. DEGS1 KO cells show elevated ROS levels, enhanced autophagy and apoptosis, impaired lysosomal integrity and mild mitochondrial dysfunction. Furthermore, whole transcriptome analysis of DEGS1 KO cells by global RNA sequencing identified impaired processes associated to neuronal differentiation and synaptic function. Morphometric quantification of neuronal features in cultured primary neurons could substantiate RNA-seq results by showing a strong reduction of neuronal complexity 24 hours after Degs1-knockdown and thus impairment of neurodevelopment. In conclusion, we here demonstrate haploid DEGS1-KO cells as a valuable in vitro model for comprehensive mechanistic studies of inherited neurodegenerative diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE213289 | GEO | 2023/01/02

REPOSITORIES: GEO

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Project description:Dysregulation of ceramide synthesis has been associated with metabolic disorders such as atherosclerosis and diabetes mellitus. Using a human hepatoma cell line (Huh7), we investigated the changes in lipid homeostasis and gene expression when the synthesis of ceramide is perturbed by knocking down serine transferases subunits 1, 2 and 3 (SPTLC123) or dihydroceramide desaturase (DEGS1). While the inhibition of serine palmitoyl transferase (SPTLC) affects ceramide production differently at the subspecies level depending upon which SPTLC subunit is silenced; depleting DEGS1 is sufficient to produce a similar outcome as knocking down all SPTLC subunits. Both the distribution of multiple lipid classes, especially at the subspecies level, and the global transcriptional profile is altered differently when either SPTLC123 or DEGS1 were silenced. The overall transcriptional changes indicate a negative regulation in biosynthetic processes and a down-regulation of genes involved in general endomembrane trafficking for both DEGS1 and SPTLC123 siRNA treated cells, but also the up-regulation of genes involved with cell migration function in DEGS1 siRNA cells. Pathway analysis indicate changes in amino acid, sugar and nucleotide metabolisms as well as vesicle trafficking between organelles occurred more robustly in DEGS1 silenced cells. Although either SPTLC123 or DEGS1 siRNA treatment positively regulated numerous genes involved with endocytosis and endosomal recycling, depleting SPTLC123 caused transcriptional changes in genes primarily involved with lipid metabolism. The alterations reflect how SPTLC or DEGS1 silenced cells respond differently to disruption in lipid flux, but also maintain cellular lipid pools through increasing endocytotic processes and down-regulating metabolic biosynthesis without developing endoplasmic reticulum stress. Also, these results are the first to demonstrate that reducing ceramide synthesis by decreasing the function of either SPTLC or DEGS1 affects cellular function differently at the level of lipid synthesis and gene expression.

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Transcriptomic analysis of HAP1 DEGS1 KO cells and their wildtype counterpart

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